The authors would like to acknowledge the "Ligue Nationale contre le Cancer" (Comit&#233; du Septentrion), the SIRIC ONCOLille (Grant INCa-DGOS-INSERM 6041) and the Institut pour la Recherche sur le Cancer de Lille (IRCL) for supporting this work. They would like to thank Delphine Taillieu and the animal facility staff for housing the mice and maintaining their welfare. We also thank Rapha&#235;lle Caillerez and Nathalie Jouy for their respective help in microscopy and flow cytometry.

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The authors declare that they have no competing financial interests.

이전 연구에서, C1498 세포주 급성 과립 5 myelomonocytic 6 또는 7 NKT 세포 백혈병 유도제로서 설명 하였다. 그러나, 문학 실증 데이터가 없거나 불완전한 중 하나였다. 여기에 제시된 프로토콜은 유동 세포 계측법, 면역, MGG 염색 및 세포 화학적 분석과 같은 다른 기술, 배양 C1498 세포를 특성화하고 주입 후 쥐에서 유도되는 백혈병의 특성을 결정하기 위해를 사용한다. 우리는 면역 염색 후 체외 배양 C1498 세포 phenotyped과 세포 계측법 분석을 수행 하였다 흐름 때 이러한 세포가 이전에 문헌 6,7에 설명 된 몇 가지 세포 표면 조혈 마커를 표현하기 때문에, 우리는 몇 가지 제한을 관찰했다. 우리의 결과와 일치, 라벨 씨 등은. 흐름 cytomet를 사용하여 C1498 세포에 성숙한 TCR의 세포 표면 발현을 관찰하지 않았다공예 염색. 그들은 CD3ε 및 TCRVβ8.2 7 mRNA를 검출 한 후 그러나 그들은 NKT 세포주로 그들을 고려. 우리는 또한 세포 (&gt; 70 %)의 대부분 TCRVβ 체인 및 CD3ε 분자의 세포 내 발현을 관찰하지만, 맥-3 분자의 수반 세포 내 발현도 있었기 때문에 자신의 조혈 계통은 확인할 수 없습니다. 마이 엘 로퍼 옥시 다제, MGG 염색 및 cytochemistry를 사용하여 기능 에스 테라 제를 분석 평가는 C1498 세포주는 골수 기원을 가지고 있었고, monoblasts 및 골수성로 구성되었음을 보여 주었다. 이러한 결과는 착색 유동 세포 계측법 분석 얻었다 맥 3 + 세포의 비율로 조화 된 있었다. 다음 단계는 키 실험을 나타내는 정량적하지 않지만 수행합니다. 사실, 그들은 전혀 또는 F를 표현 조혈 세포의 혈통과 분화 단계의 특성에 가장 적합한 기존의 방법은 지금까지 남아EW 특정 표현형 마커. 염색 유동 세포 계측법은 C1498 세포를 정맥 주사 한 후 congenic 마우스의 급성 백혈병의 개발을 설명하는 데 도움이되었다. 말초 혈액 및 다양한 기관에이 침투 CD45.2 + C1498 세포 격리했고, 그 주파수를 측정 하였다. 정량은 면역 표현형 후 고유 골수 및 비장 세포를 분석 하였다. 그들은 약간 조혈 마커를 나타낸 상기 C1498 세포 표현형 기관에서 조사 될 때 한계가 발견되었다 (이들 중 단지 소수의 B220 +이었다). 상기 관찰 급성 백혈병의 자연, 월 - 그룬 왈드 김사 염색 및 단핵구의 활동의 분석을 정의하고 과립 에스 테라 제 골수를 사용하여 수행 하였다. 결과는 C1498 세포가 myelomonocytic 백혈병의 발병을 공개, 그들의 myeloblastic 및 monoblastic 형태와 기능을 보존 것으로 나타났다. <p claspH의 오류가 결과의 잘못된 해석으로 이어질 수 있기 때문에 세포 화학적 반응과 MGG 염색을 수행 할 때이 프로토콜에서 설명하는 중요한 단계에 대한 고려의 = &quot;jove_content은&quot;&gt; 특히주의 pH를 부여해야합니다. 과립구 및 림프구는 또한 더 높은 pH 값에서이 테스트에 대한 양성 염색 할 수 있기 때문에 예를 들면, α-나프 부티레이트 에스 테라 제 활성은 단지 6.0의 pH에서 단핵 세포에 특이. 세포를 집착하는 것은 MGG 염색을 수행하기 전에 권장, 우리는 C1498 세포를 이용하여 에스 테라 제를 세포 화학적 반응을 수행하는 경우에만 CAF 고정은 만족스러운 결과를 제공하는 것으로 나타났다되지 않습니다. 계측법은 시료 (예, 혈액, 골수, 비장 세포)의 모든 과정 동안 얼음에 보관해야 상기 CD115 분자의 발현과 흐름에 의해 그 검출을 보존. 전혀 오염은 유동 세포 계측법 / 또는 면역 실험 항체의 참조 스토리지 재 관찰되지 않으면표창 및 희석 확인해야합니다. 재료 / 장비 테이블에 지정된 참조는 유동 세포 계측법 또는 면역 응용 프로그램으로 선정되었습니다. 일차 / 이차 항체 또는 공액 형광 인해 부적절한 저장 그들의 활동을 분실했을 수 있습니다 (예를 들어, 빛이나 열에 노출), 부적절한 희석, 광범위한 동결 / 해동 또는 오염 된 버퍼의 사용. 그들이 제대로 작동하는지 확인하기 위해 긍정적 인 컨트롤을 실행합니다. 마우스 골수 또는 관심의 단백질을 발현하는 것으로 알려져있다 비장 유래 세포를 사용합니다. 높은 배경이 아닌 특정 얼룩을 방지하기 위해, 세포가 제대로 세척하고 (면역에 대한) 높은 습도 유지 및 지침에 따라 항체가 희석되어 있는지 확인하십시오. 정확한 샘플의 배경 레벨을 결정하기 위해 이소 타입 대조군 항체와 차 항체에 대해 동일한 농도의 희석을 사용한다. 에스 테라 cytochemistry 실험 들면,시약 정제 마우스 비장 과립 (Ly6G +) 및 단핵구 (CD115 +) 세포를 함유하는 양성 및 음성 대조군 슬라이드를 사용하여 테스트 될 수있다. 이 연구에 기술 된 절차 C1498 세포의 주입 후 쥐에서 관찰 백혈병 많은 기능 인간 급성 백혈병 myelomonocytic 11,12와 공통 특징을 공유하는 것으로 나타났다. 침략 백혈병 세포는 성숙과 미성숙 (전구 세포 및 전구 물질) 골수 조혈 세포의 감소 결과. 단핵 세포만큼 C1498 세포는 말초 혈에서 고주파 (&gt; 20 %)에 존재한다. 비대와 비장은 백혈병 세포의 침투에 기인하는 것으로 관찰되었고, B 림프구 및 골수 세포에서 유의 한 증가는 비장 함께 관찰되었다. 혈소판 번호가 혈액 분석을 사용하여 추정 될 때 Thrombopenia도 관찰되었다. 그것은 쇼였다N, 시험 관내 실험에서 사용 C1498 세포를 용해 요소 (13)를 분비하여 정상 쥐의 조혈을 억제하는 것이다. 여러 종양 마우스 모델에서 (단핵구 및 과립 세포 포함) 미숙 골수 세포는 또한 항 - 종양 특이 적 T 세포 활성화 및 증식 (14)을 억제 비장에 골수 마이그레이션하는 것으로 나타났다. 따라서, 골수에서 관찰 한 조혈 세포의 감소는 조혈 및 / 또는 이민의 결핍 중 하나에 기인 할 수 있었다. 후자의 메커니즘은 말초 혈액에서 monocytosis의 존재 또는 비장 확대 골수성 분획의 관찰을 설명 할 수있다. 이들 세포는 조혈 개선 비장으로부터 유래 된 수 있다고도 생각할 수있다. 성숙한 B의 전구체 15 림프구 실제로, 정상 조건 하에서, 비장 B 세포의 일부 서브 세트는 확인되었다. 또한, 염증성 조건 하에서 메드ullary 조상 줄기 세포는 성숙한 단핵구 (16)의 생산을 유도하는 비장 이주 도시되었다. 이 프로토콜은 우리가 백혈병의 개발에 참여하고 있으며, 추가 기능뿐만 아니라 분자 분석이 이렇게하기 위해 사용되어야하는 메커니즘에 관한 결론을 도출 할 수 없습니다. 그러나, 이러한 데이터는 급성 myelomonocytic 백혈병의 임상 적 특징에 대한 자세한 정보를 포함하고 평가 및 새로운 치료제의 효과를 이해하는 연구자 도움이 될 것입니다.

급성 골수성 백혈병 (AML)의 성숙의 다른 단계에서 차단되어 골수 조혈 세포의 억제되지 않는 증식을 특징으로한다. 이 조절 곤란은 과립, 단핵구, 적혈구 또는 megaryocytic 분화 경로 1에 영향을 줄 수 있습니다. AML 세포는 thrombopenia, 림프구 감소증과 빈혈을 초래 장애인 조혈, 선도, 골수에 축적. 백혈병 세포는 혈액 및 비 림프 기관 침입. C1498 마우스 모델은 문헌 혈액으로 침입 설명 암세포 1941에서 백혈병 10 개월 된 C57BL / 6 (H-2 b) 암컷 마우스로부터 분리 된 이후 급성 백혈병의 모델로서 수십 년 동안 사용되어왔다 조혈 기관 (예를 들어, 비장과 림프절) 및 비 조혈 기관 (예를 들어, 간은, 폐, 난소, 신장)이 높은 증식 C1498 세포로는은에서를 통해 주입 된 후travenous, 취약 마우스 2-4로 피하 또는 복강 내 경로. 그러나,이 마우스 모델은 하나 또는 과립 2,5- myelomonocytic 6 백혈병을 유도하는 것으로보고되었다. 최근에는 2002 년에 발표 된 연구는 쥐 NKT 세포 백혈병 (7)과 같은 암의 유형을 설명했다. 따라서, 상기 문헌은 본 C1498 세포주의 특성 및이 마우스에서 유도 관련 백혈병 관한 다르다. 70 년대 - 이러한 불일치는 세부의 부족과 세포에 대한 업데이트 게시 된 정보 및 많은 연구가 1950 년에 수행 되었기 때문에 일반적으로 백혈병 질병에 주로 기인한다. 여기에서는 C1498 세포를 특성화하고 마우스에 정맥 내 주사에 의해 그들의 유도 백혈병 질병의 특성을 분석하는 방법을 설명하는 세부적인 프로토콜을 제공한다. 이 프로토콜의 첫 번째 섹션은 체외에서 배양 된 C1498 세포의 설명에 최선을 다하고 있습니다. 형광 방지표면 및 세포 내 조혈 마커에 대해 감독 기관은 유동 세포 계측법 사용하여 표현형을 결정하는 데 사용되었다. 마이 엘 로퍼 옥시 다제의 존재가 면역 형광 현미경을 사용하여 평가하고, 그 리니지 조혈 및 분화 단계는 에스 테라 제의 활성을 평가하기 cytochemistry를 이용하여 평가하였고, 5-6의 Grunwald 김사 염색을 수행 하였다. C1498 세포는 생쥐에 주입하고, 유도 된 급성 백혈병 질환이 원고의 두번째 섹션에 설명되어있다. 동일한 기술은 주파수와 골수 백혈병 및 고유 세포, 말초 혈, 비장 및 비 - 조혈 기관 (간 및 폐)의 표현형을 결정하는 데 사용 하였다. 이 프로토콜은 높은 재현성이며, 여기에 제시된 데이터는 연구자들이 새로운 치료 전략의 효과를 평가하는데 도움이 될 것이다. 이러한 백혈병 마우스 모델 이미 면역이 접근 테스트하는 데 사용 된차 다른 암 화학 요법 약물 8,9. 효능은 종양 부담과 생존율의 진화를 확인하여 평가 하였다. 이 프로토콜은 치료 중 백혈병 및 다른 조혈 세포 집단의 생존 및 분포에 대한 추가 정보를 제공하는데 사용될 수있다.

<table><tbody><tr><td>C1498 cell line</td><td>ATCC</td><td>TIB 49</td><td></td></tr><tr><td>C57BL/6J-Ly5.1</td><td>Charles River</td><td>B6.SJL-Ptprc a Pep3 b/BoyCrl</td><td></td></tr><tr><td>&lt;strong&gt;Cells culture reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>2-Mercaptoethanol, Gibco</td><td>ThermoFisher Scientific</td><td>21985</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>ThermoFisher Scientific</td><td>10270</td><td></td></tr><tr><td>HEPES, Gibco (1 M)</td><td>ThermoFisher Scientific</td><td>15630</td><td></td></tr><tr><td>Non-Essential Amino Acids Solution, Gibco&amp;nbsp;&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>11140</td><td></td></tr><tr><td>Penicillin-Streptomycin, Gibco&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>15140</td><td></td></tr><tr><td>RPMI 1640 Medium (Gibco, GlutaMAX Supplement)</td><td>ThermoFisher Scientific</td><td>61870</td><td></td></tr><tr><td>Sodium Pyruvate, Gibco (100 mM)</td><td>ThermoFisher Scientific</td><td>11360</td><td></td></tr><tr><td>&lt;strong&gt;Flow cytometry staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>anti-mouse B220 APC (1)</td><td>ebiosciences</td><td>17-0452</td><td>clone RA3-6B2, final dilution 1/100</td></tr><tr><td>anti-mouse B220 biotin (2)</td><td>ebiosciences</td><td>13-0452</td><td>clone RA3-6B2, 1/400</td></tr><tr><td>anti-mouse CD3 eFluor450 (1)</td><td>ebiosciences</td><td>48-0032</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE (2)</td><td>BD biosciences</td><td>555275</td><td>clone 17A2, 1/100</td></tr><tr><td>anti-mouse CD3 PE-Cy5 (3)</td><td>ebiosciences</td><td>15-0031</td><td>clone 145-2C11, 1/100</td></tr><tr><td>anti-mouse CD4 APC (1)</td><td>ebiosciences</td><td>17-0041</td><td>clone GK1.5, 1/500</td></tr><tr><td>anti-mouse CD4 PE (2)</td><td>ebiosciences</td><td>12-0041</td><td>clone GK1.5, 1/200</td></tr><tr><td>anti-mouse CD8 biotin (1)</td><td>ebiosciences</td><td>13-0081</td><td>clone 53-6.7, 1/100</td></tr><tr><td>anti-mouse CD8 eFluor450 (2)</td><td>ebiosciences</td><td>48-0081</td><td>clone 53-6.7, 1/500</td></tr><tr><td>anti-mouse CD11a biotin</td><td>ebiosciences</td><td>13-0111</td><td>clone M17/4, 1/100</td></tr><tr><td>anti-mouse CD11b PE&amp;nbsp;</td><td>ebiosciences</td><td>12-0112</td><td>clone M1/70, 1/200</td></tr><tr><td>anti-mouse CD16/32 biotin</td><td>ebiosciences</td><td>13-0161</td><td>clone 93, 1/400</td></tr><tr><td>purified anti-mouse CD16/32 (FcR blocking)&amp;nbsp;</td><td>BD biosciences</td><td>553141</td><td>clone 2.4G2</td></tr><tr><td>anti-mouse CD18 biotin (1)</td><td>ebiosciences</td><td>13-0181</td><td>clone M18/2, 1/100</td></tr><tr><td>anti-mouse CD18 FITC (2)</td><td>ebiosciences</td><td>11-0181</td><td>clone M18/2, 1/50</td></tr><tr><td>anti-mouse CD19 PE</td><td>ebiosciences</td><td>12-0193</td><td>clone 1D3, 1/200</td></tr><tr><td>anti-mouse CD21/35 PE</td><td>ebiosciences</td><td>12-0211</td><td>clone 8D9, 1/50</td></tr><tr><td>anti-mouse CD31 PE</td><td>ebiosciences</td><td>12-0311</td><td>clone 390, 1/100</td></tr><tr><td>anti-mouse CD34 eFluor660</td><td>ebiosciences</td><td>50-0341</td><td>clone RAM34, 1/20</td></tr><tr><td>anti-mouse CD44 PE</td><td>BD biosciences</td><td>553134</td><td>clone IM7, 1/50</td></tr><tr><td>anti-mouse CD45.2 FITC&amp;nbsp;</td><td>ebiosciences</td><td>11-0454</td><td>clone 104, 1/100</td></tr><tr><td>anti-mouse CD49b/Pan NK PE</td><td>&amp;nbsp;BD biosciences</td><td>553858</td><td>clone DX5, 1/50</td></tr><tr><td>anti-mouse CD80 biotin</td><td>ebiosciences</td><td>13-0801</td><td>clone 16-10A1, 1/200</td></tr><tr><td>anti-mouse CD86 biotin</td><td>ebiosciences</td><td>13-0862</td><td>clone GL1, 1/200</td></tr><tr><td>anti-mouse CD107b (Mac-3) PE</td><td>ebiosciences</td><td>12-5989</td><td>clone M3/84, 1/40</td></tr><tr><td>anti-mouse CD115 PE</td><td>ebiosciences</td><td>12-1152</td><td>clone AFS98, 1/100</td></tr><tr><td>anti-mouse CD117 eFluor450</td><td>ebiosciences</td><td>48-1171</td><td>clone 2B8, 1/100</td></tr><tr><td>anti-mouse CD127 PE</td><td>ebiosciences</td><td>12-1271</td><td>clone A7R34, 1/100</td></tr><tr><td>anti-mouse CD150 APC</td><td>ebiosciences</td><td>17-1501</td><td>clone 9D1, 1/20</td></tr><tr><td>anti-mouse CD274 (PD-L1) biotin</td><td>ebiosciences</td><td>13-5982</td><td>clone MIH5, 1/200</td></tr><tr><td>anti-mouse Ly-6A/E (Sca-1) PE</td><td>ebiosciences</td><td>12-5981</td><td>clone D7, 1/100</td></tr><tr><td>anti-mouse Ly-6C APC</td><td>ebiosciences</td><td>17-5932</td><td>clone HK1.4, 1/200</td></tr><tr><td>anti-mouse Ly-6G&amp;nbsp; (Gr-1) biotin (1)</td><td>ebiosciences</td><td>13-5931</td><td>clone RB6-8C5, 1/400</td></tr><tr><td>anti-mouse Ly-6G FITC (2)</td><td>ebiosciences</td><td>11-9668</td><td>clone 1A8, 1/50</td></tr><tr><td>anti-mouse MHC class I (H-2Db) biotin</td><td>ebiosciences</td><td>13-5999</td><td>clone 28-14-8, 1/50</td></tr><tr><td>anti-mouse MHC class II (I-A/I-E) PE-Cy5</td><td>ebiosciences</td><td>15-5321</td><td>clone M5/114.15.2, 1/1000</td></tr><tr><td>anti-mouse NK1.1 PE</td><td>BD biosciences</td><td>557391</td><td>clone PK136, 1/50</td></tr><tr><td>anti-mouse TCRVb FITC</td><td>BD biosciences</td><td>553170</td><td>clone H57-597, 1/50</td></tr><tr><td>streptavidin PE-Cy5</td><td>ebiosciences</td><td>15-4317</td><td>&amp;nbsp;&amp;nbsp; 1/200</td></tr><tr><td>Immunofluorescence staining reagents</td><td></td><td></td><td></td></tr><tr><td>Anti-mouse myeloperoxidase (heavy chain antibody)&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>sc-16129</td><td>1/10 (20 &amp;micro;g/ml)</td></tr><tr><td>&amp;nbsp;anti-goat IgG (Texas Red coupled antibody)</td><td>Jackson Immunoresearch</td><td>705-076-147</td><td>1/250</td></tr><tr><td>Normal donkey serum</td><td>Jackson Immunoresearch</td><td>017-000-001</td><td></td></tr><tr><td>Hoechst solution</td><td>BD Biosciences</td><td>561908</td><td>1/1000</td></tr><tr><td>Mounting medium 1 (Fluoromount)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>F4680</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>&lt;strong&gt;Esterase cytochemical staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Naphtol AS-D chloroacetate solution</td><td>Sigma-Aldrich</td><td>911</td><td></td></tr><tr><td>Dye solution (Fast Red Violet LB Base solution)</td><td>Sigma-Aldrich</td><td>912</td><td></td></tr><tr><td>pH6.3 buffer concentrate (TRIZMAL)</td><td>Sigma-Aldrich</td><td>913</td><td></td></tr><tr><td>Sodium nitrite solution</td><td>Sigma-Aldrich</td><td>914</td><td></td></tr><tr><td>Citrate solution</td><td>Sigma-Aldrich</td><td>915</td><td></td></tr><tr><td>Hematoxylin solution</td><td>Sigma-Aldrich</td><td>GHS3</td><td></td></tr><tr><td>Acetone</td><td>VWR&amp;nbsp;</td><td>20066</td><td></td></tr><tr><td>Formaldehyde solution, 37%</td><td>Sigma-Aldrich</td><td>F1635</td><td></td></tr><tr><td>&amp;alpha;-naphthyl butyrate solution</td><td>Sigma-Aldrich</td><td>1801</td><td></td></tr><tr><td>Phosphate buffer solution</td><td>Sigma-Aldrich</td><td>1805</td><td></td></tr><tr><td>Sodium nitrite Tablet</td><td>Sigma-Aldrich</td><td>1809</td><td></td></tr><tr><td>Pararosaniline solution</td><td>Sigma-Aldrich</td><td>1804</td><td></td></tr><tr><td>Methylene blue solution</td><td>Sigma-Aldrich</td><td>1808</td><td>1/10</td></tr><tr><td>mounting medium 2 (Clearmount)</td><td>Invitrogen</td><td>00-8010</td><td></td></tr><tr><td>&lt;strong&gt;May-Gr&amp;uuml;nwald Giemsa staining reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>May-Gr&amp;uuml;nwald solution</td><td>RAL Diagnostics</td><td>320070</td><td></td></tr><tr><td>Giemsa R solution&amp;nbsp;</td><td>RAL Diagnostics</td><td>320310</td><td>&amp;nbsp;&amp;nbsp; 1/20</td></tr><tr><td>pH 6.8 buffer solution</td><td>RAL Diagnostics</td><td>330368</td><td></td></tr><tr><td>&lt;strong&gt;Others materials, reagents and equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Ketamine 1000 (100 mg/ml)</td><td>VIRBAC</td><td>3597132111010</td><td></td></tr><tr><td>Xylazine SEDAXYLAN (20 mg/ml)</td><td>CEVA</td><td></td><td></td></tr><tr><td>Bovin albumin serum (BSA) powder</td><td>ThermoFisher Scientific</td><td>BP671</td><td></td></tr><tr><td>PBS solution (1x concentrate)</td><td>ThermoFisher Scientific</td><td>14190</td><td></td></tr><tr><td>Paraformaldehyde (PFA)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Saponin&amp;nbsp;</td><td>Sigma-Aldrich</td><td>47036</td><td></td></tr><tr><td>Ultra Pure 0.5 M EDTA solution, pH 8.0</td><td>ThermoFisher Scientific</td><td>15575</td><td></td></tr><tr><td>Separating solution (Pancoll Mouse)</td><td>PANBIOTECH</td><td>P04-64100</td><td></td></tr><tr><td>Lysis buffer (Red Blood Cells Lysing Buffer) (10x)</td><td>BD Biosciences</td><td>555899</td><td>1/10</td></tr><tr><td>NaOH 10 N</td><td>ThermoFisher Scientific</td><td>SS267</td><td></td></tr><tr><td>Trypan blue solution (0.4 %)</td><td>ThermoFisher Scientific</td><td>15250-061</td><td>1/2</td></tr><tr><td>50 ml tube (Falcon)</td><td>Fisher Scientific</td><td>14-432-22</td><td></td></tr><tr><td>70 &amp;micro;m Cell Strainer (Falcon)&amp;nbsp;</td><td>Corning Life Sciences</td><td>352350</td><td></td></tr><tr><td>&amp;nbsp;Chamber &amp;amp; filter card (EZ Cytofunnel Shandon)</td><td>Thermo Scientific</td><td>A78710003</td><td></td></tr><tr><td>Microscope Cover Glasses, 24 x 24mm</td><td>Knittel Glass</td><td>VD1 2424 Y100</td><td></td></tr><tr><td>Slides (Starfrost - ground edges 90)</td><td>Knittel Glass</td><td>VS1137# 077FKB</td><td></td></tr><tr><td>EDTA Tube</td><td>Greiner Bio-One</td><td>454034</td><td></td></tr><tr><td>Pasteur Pipette 150 mm (capillary tube)</td><td>Fisherbrand</td><td>1154-6963</td><td></td></tr><tr><td>26 G needle</td><td>Terumo</td><td>NN-2613R</td><td></td></tr><tr><td>&amp;nbsp;insulin syringe and needle 29 G</td><td>Terumo</td><td>BS05M2913</td><td></td></tr><tr><td>30 G needle&amp;nbsp;</td><td>Becton &amp;amp; Dickinson</td><td>304000</td><td></td></tr><tr><td>Flow cytometry tubes (blue)</td><td>Beckman Coulter</td><td>2523749</td><td></td></tr><tr><td>Water-repellent pen (Dakopen)</td><td>Dako</td><td>S200230</td><td></td></tr><tr><td>Sharp sterile scissors&amp;nbsp;</td><td>Nessi-care</td><td>SCI-01</td><td></td></tr><tr><td>Sterile forceps</td><td>Dominique Dutscher</td><td>956506</td><td></td></tr><tr><td>Thoma cell counting chamber</td><td>VWR</td><td>631-0397</td><td></td></tr><tr><td>Petri Dishes (Fisherbrand Plastic)</td><td>ThermoFisher Scientific</td><td>S33580A</td><td></td></tr><tr><td>Microcentrifuge tube (1.5 ml)</td><td>ThermoFisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>Cylindrical Restrainer 15 - 30 gm</td><td>Stoelting</td><td>51338</td><td></td></tr><tr><td>Shandon Cytospin 3 Cytocentrifuge</td><td>ThermoFisher Scientific</td><td></td><td></td></tr><tr><td>10 ml syringe</td><td>Terumo</td><td>SS-10L</td><td></td></tr><tr><td>1 ml syringe</td><td>Terumo</td><td>SS-01T</td><td></td></tr><tr><td>Ethanol</td><td>Merck</td><td>1.08543</td><td></td></tr><tr><td>Sterile gauze sponges</td><td>URGO</td><td>501580</td><td></td></tr></tbody></table>

a detailed protocol for characterizing the murine c1498 cell line and its associated leukemia mouse model

1941이 주사 급성 백혈병의 개발 결과 때문에 동계 또는 congenic 마우스에 C1498 세포의 정맥 내 주사가 수행되었다. 그러나,이 병의 성격이 잘 문헌에 기록되지 않았습니다. 여기서는 C1498 시험 관내 세포의 특성과 생체 내에서 유도 성 백혈병의 특성을 결정하기위한 프로토콜 기술을 제공한다. 이 절차의 첫 번째 부분은 조혈 리니지 배양 C1498 세포의 분화 단계를 결정하는 단계에 초점을 맞추고있다. 이를 위해, 다중 파라미터 유세포 염색 조혈 세포 마커를 검출하기 위해 사용된다. 면역 형광 현미경 cytochemistry 및 5 월의 Grunwald 김사 염색 한 다음, 각각의 마이 엘 로퍼 옥시 다제 발현 및 세포 형태 에스 테라 제의 활성을 평가하기 위해 수행된다. 이 프로토콜의 두 번째 부분에 유도되는 백혈병 질환을 설명하기 위해 최선을 다하고 있습니다생체. 후자는 혈액에서 백혈병 및 고유 셀의 주파수를 결정함으로써 달성 될 수있다 계측법 특정 염색을 이용하여 유동 조혈 기관 (예, 골수, 비장) 및 비 림프계 조직 (예, 간, 폐)을 분석한다. 백혈병의 특성은 골수 특정 에스 테라 제에 대한 5-6의 Grunwald 김사 염색 얼룩을 사용하여 확인된다. 여기, 우리는 연령대 C1498- 및 PBS-주입 된 쥐에서이 프로토콜을 사용하여 얻었다 결과를 제시한다.

C1498 마우스 모델의 특성을하기 위해, 우리는 두 가지 주요 단계를 계속했다. 우선, C1498 세포는 시험 관내에서의 혈통 조혈 성숙 단계 (도 1)을 결정하는 것을 특징으로 하였다. 이들 세포를 congenic 생쥐에 주입하고, 유기 백혈병 질환의 성질은 상이한 특징을 결정하는 평가 하였다 : 백혈병 세포의 침윤, 그 표현형, 골수의 조혈 세포의 정량화 (성숙 및 선조 / 전구체) 주파수를 C1498 세포와 혈액에 성숙 조혈 세포 및 장기 부종의 평가 (비장, 간, 폐) 및 세포 조성물. 시험 관내 C1498 세포 표현형을 특성화하기 위해, 세포를 조혈 전구체 및 성숙한 세포 (표 1)에 의해 표현되는 분자에 대한 항체로 표지하고, 그 결과 유동 cytomet를 사용하여 분석 하였다공예. C1498 세포 맥-1 (CD11b를 / CD18) (~ 7 %), B220 (&gt; 25 %)의 세포 표면 발현을위한 양성이었고, 그들은 CD3ε, T 세포 수용체 (TCR) Vβ 쇄 맥의 세포 내 발현을 나타 -3 (도 2A와 B). 세포는 세포 표면 마커 Ly6G, Ly6C, CD115, CD21 / CD35, CD19, CD3, CD4, CD8, NK1.1 및 팬 NK 분자를 부정적이었고, CD4와 CD8의 세포 내 발현 (데이터 도시하지 않음) . 이들은 다음의 조혈 줄기 세포 및 전구 세포 (표 1)의 마커에 대해 조사 하였다. 그들은 또한 CD117, CD34, 무서-1, CD150 및 CD16 / 32의 세포 표면 발현을위한 음성이었다 (데이터는 보이지 않음). 이러한 백혈병 세포를 접착 항원 제시 및 공동 자극 분자의 발현을 확인하기 위해 테스트 하였다. 세포 표면 마커 LFA-1 (CD11a에 / CD18), CD44, CD31 (PECAM-1) 및 H-2D B를 발현시키고, CD80, CD86 및 CD274 (데이터 미도시) MHC 클래스 II 부정적이었다. C1498 세포 therefore 골수 (맥-1, 맥-3) 및 림프 마커 (B220, CD3, TCR) 모두를 표명했다. 더 잘 조혈 혈통을 특성화, 마이 엘 로퍼 옥시 다제 발현은 면역 형광 현미경을 사용하여 평가 하였다. 모든 세포는 골수 유래 (도 3A)을 검증 마이 엘 로퍼 옥시 다제, 양성이었다. 세포의 대부분은 또한 α 나프 틸 부티레이트 에스 테라 제 (그림 3B, 왼쪽 패널)에 대한 긍정적 인 염색, 그들 중 일부는 나프톨 AS-D 클로로의 에스 테라 제 (검은 색 화살표) (그림 3B, 오른쪽 패널)에 대한 스테인드. 그 결과 세포가 단핵 세포 및 과립구의 혼합물을 함유하는 것으로 나타낸다. 5-6의 Grunwald 김사 염색을 수행 한 후, C1498 세포는 높은 nucleo - 세포질 비율 핵 3-5 핵소체하는 핵 주변 할로, 다수의 액포와 호 염기성 세포질 (도 3c를 폭발과 같은 형태를 표시하는 관찰 ). 일우리는 C1498 세포주 monoblasts 및 골수성 구성된다. C1498 세포 (CD45.2 +)은 다음 정맥 CD45.1 + 마우스에 주사 하였다. 쥐 세포를 주입하고 17 내지 19 일 후에 굴복. 그들은 질병 사망 전에 백혈병 유형을 분석 할 수 있도록 상기 마우스들을 희생시켰다. PBS 주입 된 대조군 마우스는, 비교를위한 동일 시점에서 분석 하였다. C1498 세포 주입 된 마우스는 5-6의 Grunwald 김사 염색 (도 4A)을 수행 한 후에 셀의 폭발과 같은 외관에 의해 입증 된 바와 같이, 그들의 골수 C1498 세포의 대량 침투를 표시. 또한 급성 myelomonocytic 백혈병의 특징이다 monoblastic 및 myeloblastic 세포의 축적을 보여주는, 자신의 단핵구 및 과립 표현형 (그림 4B 및 C)를 보존. (D)에골수 조혈 세포 번호, 백혈병 세포의 침윤, CD45.2 + C1498 세포, B의 림프구, 단핵구 및 (조상, 전구체 및 성숙 세포를 포함) 과립 인구 다음 낮았다 면역 염색 및 세포 계측법 분석 다중 파라 메트릭 흐름을 사용하여 정량화되었는지 etermine. 백혈병 세포가 조혈 세포의 36 %로 (16)로 표시 (데이터는 보이지 않음). 다른 세포 유형 모두 과립 세포와 평균 3 배에 대한 평균 4 배, B 세포 하위 집합에 대한 평균 5 배 (PBS를 주입 쥐에 비해 C1498 주입 된 쥐에서 상당히 낮은 숫자에 제시 하였다 C에 단핵구 부분 집합에 대한) (그림 5A). 백혈병 및 제어 마우스 혈액의 단핵 세포의 빈도에 대한 조사는 림프구 (도 6A)의 비교 백분율하지만 단핵구 높은 주파수를 함유하는 것으로 나타났다및 백혈병 세포. 이러한 특성은 급성 백혈병 myelomonocytic (11) (도 6b)을 대표한다. 급성 myelomonocytic 백혈병 (12)의 다른 기능 중, C1498 주입 마우스 부어 간 (간 비대), 폐와 비장 (비장) (도 7a)에 발표했다. CD45.2 + C1498 세포의 다양한 주파수는 면역 형광 염색법을 사용하여 이러한 기관에서 검출 및 분석 (그림 7B) 유동 세포 계측법했다. 로 비장 비대 침투 단핵 세포의 높은 숫자에서 발생할 수 있습니다, 우리는 또한 비장 인구의 비율을 추정했다. 상기 B 림프구, 단핵 세포 및 과립구 세포 분획의 수는 2 배의 평균 상당히 컸다 2.5 배 및 3 배 각각 제어 비장 (도 7C)보다 백혈병 비장있다. 얇은 페이지 = &quot;1&quot;&gt; <img alt="그림 1" src="/files/ftp_upload/54270/54270fig1.jpg" /> 체외 배양 C1498 셀 라인과 급성 백혈병의 생체 설명에 특성화를위한 설정 프로토콜. 조혈 계통 및 조직 배양 C1498 세포의 분화 단계의 그림 1. 도식 표현 먼저 결정 하였다. C1498 세포는 급성 백혈병의 개발을 유도 congenic 마우스에 주사 하였다. 골수, 말초 혈, 비장, 간, 폐 조직의 분리는 C1498 세포 침윤 후 주파수 표현형 및 형태 학적 변화를 결정하기 위해 수행되었다. IV : 정맥 MGG :. 월 - 그룬 왈드 김사 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <html> 광고 / 54270 / 54270fig2.jpg &quot;/&gt; 체외 문화. 도트 플롯과 세포 표면 (A) 및 세포 (B)의 히스토그램 세포 계측법 대표 흐름 후 C1498 세포의 그림 2. 표현형 분석 조혈 성숙한 세포 분화와 관련 된 분자가 표시됩니다 C1498-표현했다. C1498 세포는 세포 표면는 CD11b, CD18 및 B220 마커 또는 아이소 타입 컨트롤 특정 하였다 형광 항체를 사용하여, 배양 물로부터 수확 된 세정 표지 하였다. 세포 염색을 위해, 세포를 고정 및 투과 화 맥 3 CD3ε 대한 항체 및 TCR (T 세포 수용체) Vβ 쇄 또는 아이소 타입 컨트롤 공통 에피토프를 사용하여 라벨링 하였다. 분석은 살아있는 세포에 게이팅 사용하여 수행 하였다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <html> ntent &quot;FO : 유지-together.within 페이지 =&quot;1 &quot;&gt; <img alt="그림 3" src="/files/ftp_upload/54270/54270fig3.jpg" /> 그림 3. 기능 및 배양 C1498 세포의 형태 학적 특성. C1498 세포 배양 물로부터 수확 현미경 슬라이드에 원심 분리 하였다. (A) 마이 엘 로퍼 옥시 다제 발현 염색 면역 형광을 사용하여 수행 하였다. (B) 세포 화학적 반응은 α-나프 부티레이트 에스테라아제 (NBE) 및 나프톨 AS-D 클로로 아세테이트 에스 테라 제를 분석 하였다 (CAE) C1498 세포에서 활동. 세포는 갈색과 붉은 보라색, 큰 세포질 과립, 각각 관찰되었다 각 레이블에 대한 긍정적 인 것으로 간주되었다. C1498 세포의 (C) 월 - 그룬 왈드 김사 (MGG) 염색. 각 염색 실험은 현미경 대물 렌즈 배율을 나타낸다. 각 이미지는 세 개의 실험을 나타낸다.large.jpg &quot;대상 =&quot;_ 빈 &quot;&gt;이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오. <img alt="그림 4" src="/files/ftp_upload/54270/54270fig4.jpg" /> 그림 4. PBS-과의 골수 모폴로지 마우스를 C1498 주입. 골수 세포는 PBS-및 C1498 세포 주입 쥐에서 분리하여 현미경 슬라이드에 원심 분리 하였다. (A) 월 - 그룬 왈드 김사 (MGG) 염색. (B ) α 나프 틸 부티레이트 에스 테라 제 (NBE) 및 클로로 에스 테라 제 (CAE) 기능이 cytochemistry를 사용하여 평가 하였다 AS-D (C) 나프톨. 패널 (A)에서, 밴드 (미숙 한) 또는 분할 (성숙) 호중구는 PBS-주입 된 쥐보다 C1498 주입 쥐의 골수에서 덜 볼 수 있습니다. 패널 B 및 C는 제어 골수에서 관찰 된 숫자에 비해 백혈병 골수 단핵 세포 및 과립구의 축적 있다는 것을 나타낸다. 모든현미경 분석은 100X 배율 목표를 사용하여 수행 하였다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 5" src="/files/ftp_upload/54270/54270fig5.jpg" /> 그림 5. PBS-과의 수질 인구의 정량 분석 마우스를 C1498 주입. 골수 세포는 PBS-및 C1498 세포 주입 쥐에서 분리 된 세포 계산을 수행 한 후 추정되었다. 다른 세포 집단의 주파수가 면역 및 세포 계측법 분석 생균 게이트 흐름 후에 측정 하였다. (A)가 B 세포의 서브 세트는에서 B 림프구 (B) 과립 세포 성숙 프로 B 세포로부터 단계에서 CD19 + B220 + 세포를 포함 전구체을 포함 CD11b를 + Ly6G + 계통, - CD3. CD115 + 및 단핵구 단계 성숙하는 전구 세포를 포함 - 차 미성숙과 성숙 과립구 (C)는 단핵구 부분 집합은 CD3로 정의 하였다. n은 7 마우스 / 그룹 및 데이터 =은 SEM ± 수단을 보여주는 히스토그램으로 표시하고 있습니다. ***, p &lt;0.0001 **, P &lt;0.01, PBS-및 C1498 주입 쥐를 비교 짝이 학생의 T- 테스트. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 6" src="/files/ftp_upload/54270/54270fig6.jpg" /> PBS-과의 단핵 세포 하위 집합의 그림 6. 혈액 분석 쥐를 C1498은 주입. 각각 PBS-및 C1498의에서 CD3 +와 B220 + 세포로 정의 하였다 (A) T와 B 림프구 비율의 도트 플롯 세포 계측법 대표 유동 세포 주입.와 CD115 + Ly6C 높은 세포 - 마우스 (B) C1498의 백혈병 및 제어에 단핵구 세포 주파수 (PBS) 마우스 CD115 + Ly6C를 분석하여 측정 하였다. 분석은 생균 게이팅에 의해 수행되었다. 백혈병 및 제어 쥐를 비교하기 위해, C1498 세포는 제외 하였다 + CD45.2입니다. <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig6large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <img alt="그림 7" src="/files/ftp_upload/54270/54270fig7.jpg" /> 백혈병 및 제어 마우스의 비장 인구의 그림 7. 평가. (A) 마우스 백혈병 부종, 간, 폐, 비장의 대표 사진은 대조군 마우스와 비교. 비장은 조직 중단 다음 수집 및 무게, 및 비장 세포를 계수 하였다되었다. (B) 히스토그램은 diff에서 백혈병 세포의 주파수를 나타내는erent 기관 면역이 CD45.2 + 세포에 대해 수행하였으며, 그 결과는 유동 세포 계측법으로 분석 한 후. (C) 비장 B, 면역 후 과립구 및 단핵구 세포 수의 추계 분석 게이팅 유동 세포 계측법 살아있는 CD19 + B220을 식별하기 위해 수행되었다 + , CD3 - CD11b를 + Ly6G +, CD3 - CD11b를 + Ly6C - 및 CD3 - CD11b를 + Ly6C 높은 세포. 폐, 비장 및 간에 대해 표시 스케일 바는 1cm를 나타냅니다. .. n은 5 = - 8 마우스 / 그룹 및 데이터 SEM ± 수단으로 막대 그래프로 표시됩니다 * P &lt;0.05, **, P = 0.0033, PBS-과 비교 짝이 학생의 T- 테스트 C1498이 주입 된 쥐 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54270/54270fig7large.jpg" target="_blank">하세요 이 그림의 더 큰 버전을 보려면 여기를 클릭하십시오.</a> <table fo:keep-together.within-page="1"><tbodY&gt; <tr><td> 셀 유형 </td><td> 막 또는 세포 내 분자 </td></tr><tr><td></td><td></td></tr><tr><td> 전구체와 성숙한 세포 </td><td></td></tr><tr><td> NK 세포 </td><td> NK1.1 +, 팬-NK의 + </td></tr><tr><td> NKT 세포 </td><td> NK1.1 +, 팬-NK +, TCR Vbeta + (8.2), CD3 + </td></tr><tr><td> T 림프구 </td><td> TCR Vbeta +, CD3 +, CD4 +, CD8 + </td></tr><tr><td> B 세포 전구체와 B 림프구 </td><td> B220 +, CD19 +, CD21 / 35 + </td></tr><tr><td> 과립 전구체 및 과립구 </td><td> Ly6G +, 맥-1 +, CD11b를 + </td></tr><tr><td> 단핵구 전구체 및 단핵구 / 대 식세포 </td><td> CD11b를 +, 맥-1 + 맥 - 3 +, CD21 / 35 +, CD115 +, Ly6Chi </td></tr><tr><td></td><td></td></tr><tr><td> 조상 </td><td></td></tr><tr><td> 능성 전구 세포 </td><td> CD117 + 무서-1+ CD34 + (선형 적 CD150-) </td></tr><tr><td> 림프 - 프라임 능성 전구 세포 </td><td> CD117hi 무서-1hi CD127 + (선형 적) </td></tr><tr><td> 일반적인 림프 전구 세포 </td><td> CD117lo 무서-1lo CD127 + (선형 적) </td></tr><tr><td> 공통 골수 전구 세포 </td><td> CD16 / 32lo CD117 + CD34int (선형 적 무서-1) </td></tr><tr><td> 과립구 - 대 식세포 선조 </td><td> CD16 / 32hi CD117 + CD34hi (선형 적 무서-1) </td></tr><tr><td> 거대 핵 세포 - 적혈구 전구 세포 </td><td> CD16 / 32lo CD117 + CD34lo (선형 적 무서-1) </td></tr><tr><td></td><td></td></tr><tr><td> 조혈 줄기 세포 </td><td> CD117 + 무서-1 + CD150 + (선형 적 CD34-) </td></tr></tbody></table> 표 조혈 세포 계통과 차별화 1. 마커. CD : 차별화의 클러스터; 린 : 성숙한 세포의 마커; 봐라:낮은 표현; 안녕하세요 : 고 발현; INT : 중간 표현; NK : 자연 살해 세포; TCR : T 세포 수용체.

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A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

이 논문은 시험 관내 및 주사 후 마우스에서 유도 된 급성 백혈병 C1498 세포 배양을 특성화하는데 사용될 수있는 기술적 인 방법을 제공한다. 표현형 및 기능 분석은 유동 세포 계측법, 면역 형광 현미경, cytochemistry 월 - 그룬 왈드 김사 염색을 사용하여 수행됩니다.

쥐 C1498 세포주와 관련 백혈병 마우스 모델을 특성화에 대한 자세한 프로토콜

The intravenous injection of C1498 cells into syngeneic or congenic mice has been performed since 1941. These injections result in the development of ...

a-detailed-protocol-for-characterizing-murine-c1498-cell-line-its

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19.9K 조회수.  INSERM, UMR-S-1172, Centre de Recherche Jean-Pierre Aubert (JPARC).  이 논문은 시험 관내 및 주사 후 마우스에서 유도 된 급성 백혈병 C1498 세포 배양을 특성화하는데 사용될 수있는 기술적 인 방법을 제공한다. 표현형 및 기능 분석은 유동 세포 계측법, 면역 형광 현미경, cytochemistry 월 - 그룬 왈드 김사 염색을 사용하여 수행됩니다. 

비디오: A Detailed Protocol for Characterizing the Murine C1498 Cell Line and its Associated Leukemia Mouse Model

Balmer Series

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit around the nucleus. Importantly, an atom cannot discharge energy while its electrons are in stationary states. An electron can only emit energy by changing energy states. To change energy states, an electron must move from one orbit to another by either absorbing or emitting energy. This change can only occur if the absorbed or emitted energy equals the difference between the two states. Electrons cannot exist between orbits.
The quantum number, n, is used to label the different energy states. The lowest energy state is the ground state, which is n equal to one. The excited states are labeled n equal to 2, 3, 4, and so on. When an electron at the ground state absorbs a photon whose energy equals the difference between the ground and second state, the electron becomes excited and transitions from the ground state to the n= 2 excited state. If the energy of the photon equals the difference between the ground and third state, the electron moves to the n=3 state.
According to Bohr&#8217;s model, the potential energy of an electron in the nth level can be calculated using the following equation:
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_1.jpg" />
where En is the potential energy, R is the Rydberg constant (1.0974 &#215; 107 m-1), h is Planck&#39;s constant (6.62607004 &#215; 10-34 m2&#183;kg/s), and c is the speed of light (~ 3 &#215; 108 m/s). Electrons can also spontaneously return to the ground state or any other lower excited state. When this happens, the excess energy is released in the form of an emitted photon. The energy of the photon is equal to the energy difference between the higher and lower energy states. That energy corresponds to wavelengths of light. Since each type of atom has different energy levels, the light emitted from each transition varies for each atom. For a sample of mixed molecules, the emitted light contains a range of wavelengths in what is called a continuous spectrum. For a sample containing atoms of a single element, the emitted light contains only certain wavelengths, which can be viewed as discrete lines once separated by a prism.
<h4>The Hydrogen Atom</h4>
Looking specifically at the hydrogen atom, the excitation of its electrons requires the absorption of sufficient energy to split the bond in the diatomic molecule H2. Since more energy is used to split the molecule than needed, the electrons in the hydrogen atom absorb the excess energy and are excited to a higher energy level. When the electrons spontaneously return to a lower energy level, light is emitted, which corresponds to the energy difference between the excited level and lower level.
When discussing the emission of energy, the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. The wavelengths of emitted light ultimately depend on the energy difference between the two levels.
In a pure sample of hydrogen gas, the emission spectrum appears as distinct lines of discrete wavelengths that are specific to the element hydrogen. Some of these lines are in the visible range of the electromagnetic spectrum, while some are in the ultraviolet or infrared range.
<h4>The Balmer Series</h4>
The series of visible lines in the hydrogen atom spectrum are named the Balmer series. This series of spectral emission lines occur when the electron transitions from a high-energy level to the lower energy level of n=2. Johann Balmer observed these spectral lines at 410.2 nm, 434.1 nm, 486.1 nm, and 656.3 nm, which correspond to transitions from the n=6, n=5, n=4, and n=3 energy levels to the n=2 level, respectively.
Balmer was able to relate these wavelengths of emitted light using the Balmer formula.
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_2.jpg" />
Here, &#955; is the observed wavelength, C is a constant (364.50682 nm), n is the lower energy level with a value of 2, and m is the higher energy level, which has a value greater than 3. This observation was then refined by Johannes Rydberg, where R is the Rydberg constant.
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_3.jpg" />
Remember that this equation describes emitted light, so the higher energy level is considered the initial level, or ni, while the lower level is considered the final level, or nf. In the case of the Balmer series, nf is equal to 2. This equation was combined with the Bohr model to calculate the energy needed to move an electron between its initial and final energy levels, &#916;E.
<img src="https://www-jove-com.bdigitaluss.remotexs.co/CDNSource/lm/labs/54/54_Concepts_4.jpg" />
Later, other spectral series for the hydrogen atom were discovered. For example, the Lyman series contains emission lines with energies in the ultraviolet region.
<h4>References</h4>
<ol>
	<li style="font-size:17px">Kotz, J.C., Treichel Jr, P.M., Townsend, J.R. (2012). Chemistry and Chemical Reactivity. Belmont, CA: Brooks/Cole, Cengage Learning.</li>
	<li style="font-size:17px">Silderberg, M.S. (2009). Chemistry: The Molecular Nature of Matter and Change. Boston, MA: McGraw Hill.</li>
</ol>

The Hydrogen Atom: Bohr Model and Balmer Series - Concept

발머 시리즈

The Bohr Model
Niels Bohr proposed a model for the hydrogen atom in 1913 that described discrete energy states are associated with a fixed electron orbit ...

교육

JoVE Lab Manual

화학

Genetics of Organisms

<h4>Mendelian Genetics</h4>
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this process as changes in the frequency of alleles at individual genetic loci. This simple representation of how evolution occurs dates to Gregor Mendel&#8217;s analysis of trait inheritance patterns in pea plants, first presented in 1865. Mendel determined, using rigorous collection of data, that noticeable traits are controlled by two alleles of each gene (he called them factors), one from each parent. In addition, he concluded that some alleles are dominant to others (deemed recessive). Interestingly, Mendel&#8217;s data was not appreciated until 1900, probably in part because his approach to the study of inheritance was so divergent from his predecessors. Even after his brilliant experiments were rediscovered, it took 30 additional years for Mendelian inheritance to become integrated with evolutionary theory and to gain universal acceptance.
Why was such a seemingly clear and straightforward process dormant for so long and then the center of controversy for decades? One key to the controversy is that we rarely see the clear patterns of inheritance described by Mendel. Animal and plant breeders most often see evidence for what was described as blending inheritance, or offspring whose phenotypes are intermediate between those of their parents, rather than the discrete segregation of traits into well-defined categories, like smooth versus wrinkled peas or red versus white flowers. Mendelian inheritance thus did not align well with practical experience and it took decades to reconcile the two. Additionally, there are now known to be modes of inheritance that, at first glance, appear to violate Mendel&#8217;s laws. Deeper investigations into genetics using molecular biology have effectively characterized these novel modes of inheritance. The new information does not invalidate Mendel&#8217;s findings, however, it simply enhances his model.
<h4>Phenomena that Violate Mendelian Rules</h4>
There are three main phenomena: epistasis, pleiotropy, and sex-linkage that appear to violate the basic dominance and recessivity rules of Mendelian inheritance. Epistasis occurs when two or more genes or alleles interact to affect a phenotype. A function of one gene product may be required to allow another gene to be expressed or to function normally. On the other hand, pleiotropy occurs when one gene controls the expression of multiple phenotypes in an individual. For example, a large proportion of cats with white fur and blue eyes are deaf 1. Pleiotropy is often antagonistic, meaning that the same gene causes beneficial changes in one facet of an individual&#8217;s phenotype while at the same time causing damaging changes in some other aspect of an individual&#8217;s phenotype. Antagonistic pleiotropy is seen as a tradeoff or constraint on evolution, as proposed by George C. Williams. For example, the age specific decline in performance, or senescence, is thought to be a pleiotropic trait controlled by the same genes that increase early-life fecundity2,3.
<h4>Sex Linkage</h4>
Sex linkage is another phenomenon discovered as a counter-example to basic Mendelian inheritance. In many organisms, pairs of sex chromosomes determine gender. In humans and many mammals, XX individuals are female while XY individuals are male. Male offspring inherit an X from their mother and a Y from their father; females inherit an X from their mother and an X from their father. In most species, the ratio of males to females at birth under &#8220;normal&#8221; environmental conditions is 1:1. Y-chromosomes undergo little recombination with X chromosomes during meiosis, due to the fact that, over evolutionary time, they have lost a substantial number of genes. Interestingly, there are only 16 genes shared by the human X and Y chromosomes4.
For X-linked traits (traits encoded by genes on the X chromosome) females can be homozygous (having two copies of the same allele) or heterozygous (having two different alleles). If the trait is recessive, females will only show the phenotype for the trait if they are homozygous. Heterozygous females &#8220;carry&#8221; the recessive allele, but do not express the phenotype of that allele. Statistically speaking, half of her sons will inherit this recessive allele and all will express the phenotype because males have only one X chromosome.
A commonly used example of an X-linked phenotype is hemophilia, a disease that prevents the synthesis of blood clotting proteins. If a female is heterozygous (is a carrier, genotype XH Xh) mates with a normal male (genotype XH Y), 50% of the sons will be affected. If the female was instead homozygous for the trait (genotype Xh Xh ), none of her daughters would be hemophiliacs but 100% of her sons would be. The differential phenotypic ratios seen in sons and daughters appears to violate Mendel&#8217;s laws, yet Mendel&#8217;s first law still applies; each offspring has a 50% chance of inheriting an X-chromosome carrying the hemophilia allele from a heterozygous mother5.
Like humans, the fruit fly Drosophila melanogaster has an XY-sex determination system. D. melanogaster make excellent laboratory organisms because they are easy to keep, breed, and manipulate. Wild-type flies have what is considered normal body morphology and red eyes. Many lines are readily available for purchase, including some with mutations resulting in flies without eyes, with eyes of varying color, or missing wings. These organisms can be used to study or demonstrate both Mendelian and non-Mendelian inheritance patterns. In fact, 1933 Nobel Prize winner, Thomas Hunt Morgan, first noticed that some genes are sex-linked using this system6.
<h4>References</h4>
<ol class="references">
	<li>Strain, George M., &#39; Cat Breeds With Congenital Deafness,&#39; [Online]. Available: https://www.lsu.edu/deafness/catbreeds.htm. [Accessed 29 August 2018].</li>
	<li>W. GC., &#39;Pleiotropy, natural selection, and the evolution of senescence.,&#39; Evolution, vol. 11, p. 398&#8211;411, 1957.</li>
	<li>T. &#38; S. P. S. Flatt, &#39;Integrating Evolutionary and Molecular Genetics of Aging,&#39; Biochimica et Biophysica Acta, vol. 1790, no. 10, p. 951&#8211;962, 2009.</li>
	<li>D. Bachtrog, &#39;Y chromosome evolution: emerging insights into processes of Y chromosome degeneration,&#39; Nature Reviews. Genetics, Vols. 14(2), 113&#8211;124, 2013.</li>
	<li>US National Library of Medicine, &#39;Hemophilia,&#39; Genetics Home Reference, [Online]. Available: https://ghr.nlm.nih.gov/condition/hemophilia. [Accessed 31 August 2018].</li>
	<li>&#39;The Nobel Prize in Physiology or Medicine 1933,&#39; [Online]. Available: https://www.nobelprize.org/prizes/medicine/1933/summary/. [Accessed 31 August 2018].</li>
</ol>

Genetics of Organisms: REF and WEF Fly Genetics - Concept

유기체의 유전학

Mendelian Genetics
Evolution is caused by changes in the genetic composition of populations. In the field of population genetics, scientists model this ...

생물학

유전학

Animal Behavior

<h4>Drosophila as a Model Organism</h4>
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly recognized as a fruit-loving pest. There are multiple reasons that make D. melanogaster an excellent experimental organism: their roughly two-week generation time allows studying multiple generations, they are easily maintained in very small tubes, and their sexual dimorphism enables investigators to easily distinguish between males that have black coloration towards the posterior and females that have banded abdomens.
Thomas Hunt Morgan and his students were among the first to make use of Drosophila, in the early 20th century. In 1933, Morgan received a Nobel Prize for his discovery of the role of chromosomes in heredity, which was founded on his observations of mutant Drosophila. Morgan continued his studies by generating new mutants with radiation. The presence of multiple mutants with discrete effects on the appearance of the fly enabled him to look at patterns of multifactor inheritance. A key aspect of his early work was the discovery of apparent violations of Mendel&#8217;s second law, which states that genes segregate independently of one another. Morgan&#8217;s observations indicated that genes will not segregate independently if they are joined together on the same chromosome. Furthermore, it became possible to determine gene locations on a chromosome relative to each other through analyses of how often they recombined thus triggering gene mapping studies to identify the location of a gene and distances between genes. Consequently, associates of Morgan&#8217;s lab received two additional Nobel Prizes for other studies of Drosophila genetics. Morgan&#8217;s success and the accumulating information about Drosophila inspired others to adopt them as experimental organisms, which added to our knowledge of them and progressively increased their value as a model organism. Other scientists have developed many mutations. Over 27,000 unique Drosophila lines are now maintained as stock cultures at the Drosophila stock centers and are readily available to laboratories that use them as tools for studying diverse aspects of biology, including behavior1.
<h4>Drosophila Ethology</h4>
Animal behavior, or ethology, is the study of how animals interact with each other and their environments. Although it is a relatively young biological field, ethology has important implications since an organism&#8217;s survival depends on how it behaves with regard to possible mates, food sources, and natural obstacles. Studying these organisms helps scientists to understand the contexts of these behaviors and the underlying mechanisms as to how they have evolved. Thus, a goal of the study of animal behavior is to find a genetic basis for the behaviors exhibited by individuals or groups of individuals. For example, kin selection, first proposed by William Hamilton, suggests that individuals will behave altruistically when making a sacrifice for a family member rather than a stranger2. This is because related individuals share more genes than unrelated ones, and the future reproduction by their relatives contributes in a way to their own fitness, or reproductive success. Hence, observing how organisms like Drosophila interact with their environment can allow us to understand how these behaviors are hard-wired. Additionally, these behaviors provide insight into how these organisms have adapted to cope with their environment and survive.
Directional behaviors are commonly studied to understand the basis of how and why an organism moves in its environment. Kinesis and taxis are two forms of directional behaviors. Kinesis involves stimulus-induced movements in random directions, such as the random movement of flies when chased away. On the other hand, taxis involves stimulus-induced movement towards a specific direction, such as moths flying towards a lightbulb. Taxis behaviors are positive if the animal moves towards the stimulus and negative if the animal moves away from the stimulus. Furthermore, taxis behaviors are named based on the specific stimulus that induces them. For example, geotaxis is a response to gravity, where positive geotaxis means that the organism moves with gravity. Phototaxis is movement in response to light, such as positive phototaxis of moths towards a lightbulb. Similarly, chemotaxis is attraction to or avoidance of an airborne chemical cue, such as the negative chemotaxis of many predators, including humans, in response to the smell of skunk spray. Furthermore, there are many other types of taxes that are studied by scientists, including aerotaxis, barotaxis, hydrotaxis, and magnetotaxis, that are movements in response to oxygen, pressure, water, and magnetic fields, respectively.
<h4>Study of Drosophila taxis behavior</h4>
A common and simple method to study Drosophila taxis behavior is to use a choice chamber, which allows the observation of a directional response of the fly to a particular stimulus, such as gravity, light, or a chemical. Drosophila are inserted in the center of the choice chamber and then allowed to wander freely about the chamber, which is designed so that alternative stimuli can be presented at opposing sides. After a period of wandering, the number of flies is counted on either side of the choice chamber. The number of flies on each side of the chamber is then analyzed with the Chi Square test to gauge whether or not there is a preference for a stimulus.
Comparison of directional behavior of mutant and wild-type Drosophila allows researchers to determine the role of the mutated gene in that specific behavior. Moreover, scientists are also trying to understand how animals decide what to do in the presence of combinations of stimuli, including conflicting stimuli3. Together, these studies can help predict how organisms will react to certain stimuli or changes in environmental factors.
<h4>References</h4>
<ol class="references">
	<li>Cook KR, Parks AL, Jacobus LM, Kaufman TC, Matthews K. New research resources at the Bloomington Drosophila Stock Center. Fly. 2010, Vol. 4, 1: 88-91.</li>
	<li>WD, Hamilton. Altruism and related phenomena, mainly in social insects. Ann Rev Ecol Systematics. 1972, Vol. 3, 193-202.</li>
	<li>Gepner R, Skanata MM, Bernat NM, Kaplow M, Gershow M. Computations underlying Drosophila photo-taxis, odor-taxis, and multi-sensory integration. eLife. 2015, Vol. 4, e06229.</li>
</ol>

Animal Behavior: Studying Taxis Behavior of Drosophila - Concept

동물 행동

Drosophila as a Model Organism
The common fruit fly, Drosophila melanogaster, is a widely used model organism in biology, though it may be more commonly ...

생태학

Hardy-Weinberg and Genetic Drift

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are therefore very difficult to measure. In general, studying phenomena like this requires the use of mathematical models which are built using parameters that can be conveniently measured. These models are then used to make predictions about how changes to the system might affect the outcome. 
For example, changes to the frequency of genetic alleles at individual loci in species are often observed over long time periods, but are not generally observable over short time periods. The use of computer models allows researchers to predict changes within a species&#8217; gene pool using observations that have already been gathered, and allow for simulations of a potentially unlimited number of generations of the population. This would certainly not be possible within a human lifetime.
<h4>The Hardy-Weinberg Equilibrium Principle</h4>
One equation used to model populations is the Hardy-Weinberg equilibrium equation. It was formulated independently in 1908 by both G. H. Hardy and Wilhelm Weinberg1,2. The simple equation describes the expected allele frequency of a population that is not evolving. Because most real-life populations are evolving in response to the forces of natural selection, this formula acts as a useful null hypothesis. It is used to test whether or not different types of selection are, or are not, occurring. If measured allele frequencies differ from those that were predicted using the Hardy-Weinberg equation, then the gene in question is undergoing evolutionary change.
The Hardy-Weinberg equation can be represented as p2 + 2pq + q2 = 1 where p represents the frequency of the dominant allele and q represents the frequency of the recessive allele for the gene in question. Each expression in the equation represents the predicted frequency of one of the three possible diploid genotypes. Homozygous dominant frequency is represented by p2, homozygous recessive by q2, and heterozygous by 2pq. If these frequencies are summed, it adds up to 1, which makes sense since the full population had been divided into the three available categories.
It is also possible to describe the population, or gene pool, in terms of the two alleles, without regard to how they are packaged into diploid individuals. This equation is represented as p + q = 1, that is, the frequencies of the dominant and recessive alleles must add up to 1 within the population. Again, this makes sense, since the model includes just two allele possibilities. Notice that the frequency of genotypes in the population is just the quadratic expansion of the frequency of alleles in the population because (p + q)2 = p2 + 2pq + q2.
The Hardy-Weinberg equation, like most other models, requires a series of assumptions. Usefully, if the real-life data differs from the predicted data, it is possible to hypothesize which of the starting assumptions was false. The assumptions of the Hardy-Weinberg equilibrium equations are: 1) the population is very large, 2) the population is closed, meaning that there are no individuals immigrating into or emigrating out of the population, 3) there are no mutations occurring on the gene in question, 4) individuals within the population are mating at random&#8212;individuals are not choosing their mates, 5) natural selection is not occurring. Again, keep in mind that the Hardy-Weinberg equilibrium equation is a null hypothesis. Frequently, real populations violate one or more of these assumptions. The equation is used to determine if and how evolution is happening.
<h4>Genetic Drift</h4>
In a relatively small population, a condition that violates the first Hardy-Weinberg assumption, it is possible for allele frequencies to have resulted from chance. This phenomenon is referred to as genetic drift. One version of this is referred to as the founder effect. If a small number of individuals move to an isolated location and start a new population, the specific genetics of those particular individuals will shape the future generations. This new small gene pool may have the same allele frequency as the original, but it is also possible, even likely, that it does not. Say the original population included 50% dominant alleles and 50% recessive. In the extreme, if all of the individuals in a migrating founder group are homozygous recessive, then the dominant allele has been lost completely and the recessive is now at 100%. A similar phenomenon, called the bottleneck effect, can occur if a population sustains a large drop in numbers due to a natural disaster, human intervention, or disease.
Importantly, genetic drift represents a type of evolution that is not necessarily adaptive. It does not specifically select for traits that are fit for the environment. Nonetheless, it is an important evolutionary force in shaping populations with a small number of individuals and will cause deviations from Hardy-Weinberg predictions. In populations that are larger, deviations from the predictions are more likely to mean that the population in question is undergoing evolution through natural selection. Under these circumstances, further consideration of the mechanisms causing this evolution can be explored.
Modern applications of the Hardy-Weinberg model include analysis of human and animal populations in terms of how their immune systems relate to their susceptibility to infectious disease3,4. Many research groups are cataloging genes which encode specific immune system molecules, such as CCR5 and the major histocompatibility complex (MHC, known in humans as the human leukocyte antigen (HLA), and correlating this information to epidemiological studies regarding disease and its progression3,5-6. From comparing these two types of data sets, patterns are emerging showing that some individuals are genetically resistant to particular infections while others are more likely to succumb to disease4. Hardy and Weinberg&#8217;s model has been essential to the analysis of this data and has contributed to our understanding of how infections have shaped evolution.
<h4>References</h4>
<ol class="references">
	<li>Hardy, G. H. (Jul 1908). 'Mendelian Proportions in a Mixed Population' (PDF). Science. 28 (706): 49&#8211;50. doi:10.1126/science.28.706.49. ISSN 0036-8075. PMID 17779291.</li>
	<li>Weinberg, W. (1908). '&#220;ber den Nachweis der Vererbung beim Menschen'. Jahreshefte des Vereins f&#252;r vaterl&#228;ndische Naturkunde in W&#252;rttemberg. 64: 368&#8211;382.</li>
	<li>Eguchi S, Matsuura M. Testing the Hardy-Weinberg equilibrium in the HLA system. Biometrics. 1990 Jun;46(2):415-26. PubMed PMID: 2364131.</li>
	<li>Phillips KP, Cable J, Mohammed RS, et al. Immunogenetic novelty confers a selective advantage in host-pathogen coevolution. Proc Natl Acad Sci U S A. 2018;115(7):1552-1557.</li>
	<li>Lim JK, Glass WG, McDermott DH, Murphy PM CCR5: no longer a 'good for nothing' gene--chemokine control of West Nile virus infection. Trends Immunol. 2006 Jul; 27(7):308-12.</li>
	<li><a target="__blank" href="https://hla-net.eu/tools/frequency-estimation/">https://hla-net.eu/tools/frequency-estimation/</a></li>
	<li>Santiago Rodriguez, Tom R. Gaunt and Ian N. M. Day. Hardy-Weinberg Equilibrium Testing of Biological Ascertainment for Mendelian Randomization Studies. American Journal of Epidemiology Advance Access published on January 6, 2009, DOI 10.1093/aje/kwn359. http://www.oege.org/software/hwe-mr-calc.html</li>
</ol>
<h4>Further Reading</h4>
Andrews, C. (2010) The Hardy-Weinberg Principle. Nature Education Knowledge 3(10):65 https://www.nature.com/scitable/knowledge/library/the-hardy-weinberg-principle-13235724

Hardy-Weinberg Equilibrium Principle and Genetic Drift - Concept

하디-와인버그(Hardy-Weinberg)와 유전적 부동(genetic drift)

Evolutionary change is interesting and important to study, but changes in populations occur over long periods of time and in huge physical spaces and are ...

진화

Evolutionary Relationships

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system was improved upon during the Renaissance and then, subsequently, by Carolus Linnaeus in the mid 1700&#8217;s. These more formal classification and organization systems grouped species by their physical similarity to one another. For example, all vertebrates have a backbone, but invertebrates do not. Traits like the backbone are called synapomorphies, which are traits that are shared by a group of organisms, presumably because they were derived from a common ancestor. As we will explore, this method has been shown to have limitations and has more recently been amended to include genetic analysis. Still, scientists construct trees called dendrograms to create a visual representation of how species are related to one another and share common ancestors. These dendrograms can aid in our understanding of the evolutionary processes that drive these relationships. Genetic comparisons have added an important tool guiding the analysis of evolutionary relationships.
<h4>Types of Dendrogram</h4>
A type of dendrogram, called a cladogram, depicts the hypothetical genealogical relationships between species with the tips (or leaves) of the tree representing a species and the branches showing how species are related to each other. A slightly more complicated type of tree, called a phylogram, differs from a cladogram in that the branches leading to the species are of different lengths. The length of a branch in this type of tree represents the degree of change between species: the longer the branch, the more time since the species have diverged from a common ancestor. In both types of tree, the common ancestor of a group of species is indicated by a node, which is the point where a series of branches meet. Species that are more closely related to each other (most recently shared a common ancestor) will be located closest to the node. The two species that share a node are called a sister group1.
<h4>Understanding Evolutionary Relationships using Genetic Data</h4>
Historically, cladograms were constructed by comparing the morphology (physical structure) of organisms. This method is still practiced but the techniques have been modernized to include comparison of DNA (deoxyribonucleic acid) sequences between species. Using DNA for building trees has several advantages over relying solely on morphology, including being able to calculate an estimate of how long ago different species shared a common ancestor1. However, using DNA is not always feasible, especially when trees include extinct organisms. DNA is best found in soft tissues, which are not preserved during the fossilization process, and therefore it is uncommon for a DNA sample of an extinct species to be available.
DNA is passed on from parents to their offspring in hereditary units called genes. The nucleotide (A, G, C, and T) sequence of genes found in different species are frequently quite similar, likely due to their having come from a common ancestor. This fact allows researchers to align sequences from different species with one another to build the trees described above. Species with more similarity between their nucleotide sequences will be placed next to each other in a tree, and species with less sequence similarity will be placed further apart from each other.
Bioinformatics are the tools used by biologists to analyze large datasets using a combination of computer science, mathematical modeling, and statistics. One such tool is called BLAST (Basic Local Alignment Search Tool), which can be used to quickly search the entire genome of any species that is available in the NCBI (National Center for Biotechnology Information) database2. The NCBI database combines several different databases that hold different types of DNA sequence information. The process of a BLAST search includes complex computer algorithms, but basically, BLAST aligns sequences of each nucleotide base from a submitted DNA sequence (known as the query sequence) with sequences in the data base that most closely match it. The DNA sequences that are found will be listed in order of similarity to the sequence in question, and will therefore be from species closely related to the species containing the query gene. This comparison may or may not depict the actual evolutionary relationship between species because genes evolve at different rates. Additionally, genomes sometimes contain more than one instance of a similar sequence.
Comparison of DNA sequences of genes is valuable beyond consideration of evolutionary relationships. Frequently, genes are identified in model organisms, such as the fruit fly, Drosophila melanogaster, or the mouse3. Integral to studying a gene, the function of its product is commonly identified and analyzed. If a researcher is interested in studying that function in a different organism (humans for example), BLAST or other bioinformatic tools can be used to find candidate genes based on their similarities to the genes of known function from model organisms.
Human genes can also be used as the starting point to find homologs in model organisms. In fact, human disease research depends heavily on this. Once a human gene of interest is identified, mice can be genetically manipulated to have the homologous gene disrupted, or &#8220;knocked out,&#8221; creating a model of the human disease that can be studied in order to understand and treat the disease. There are many of these mouse strains currently available. For example, there is a mouse model for human Cystic Fibrosis (CF) called the Cftr knockout mouse and another modeling atherosclerosis, called the Apoe knockout3. 
 
<h4>References:</h4>
<ol class="references">
	<li>Annenberg Learner. Online Textbook: Unit 3 Evolution and Phylogenetics. Rediscovering Biology. [Online] 2017. [Cited: August 23, 2018.] https://www.learner.org/courses/biology/textbook/compev/compev_3.html.</li>
	<li>National Center for Biotechnology Information, U.S. National Library of Medicine. BLAST: Basic Local Alignment Search Tool. [Online] https://blast.ncbi.nlm.nih.gov/Blast.cgi.</li>
	<li>National Human Genome Research Institute. Background on Mouse as a Model Organism. National Human Genome Research Institute. [Online] December 2002. [Cited: August 23, 2018.] <a href="https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/" target="__blank">https://www.genome.gov/10005834/background-on-mouse-as-a-model-organism/</a>.</li>
</ol>

Evolutionary Relationships: Using BLAST to Test Evolutionary Hypotheses - Concept

진화적 관계

Humans have been attempting to properly classify living things since Aristotle made the first attempt during the 4th century BC. Aristotle&#8217;s system ...

Animal Diversity

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular eukaryotes that move, consume organic matter, and reproduce sexually. Although these attributes are shared, species within this kingdom are also extremely diverse. This diversity is due to adaptation of each species to a different niche. The niche of a species includes the area, function, and interrelationship of that species with other biotic and abiotic factors in its environment. Niche specialization through evolutionary adaptation allows species to survive and reproduce effectively in their environment and reduces competition among species within the same habitat1-2.
<h4>Classification</h4>
Diversity in Kingdom Animalia has led to the classification of 36 distinct phyla, based on their evolutionary lineage. Seven of these phyla will be discussed, including Porifera, Cnidaria, Platyhelminthes, Annelida, Mollusca, Arthropoda, and Chordata. The first, phylum Porifera, is the earliest, simplest, most ancestral phylum. It includes multicellular, asymmetrical filter feeding sponges that lack distinct tissue layers. Interestingly, sponges can regrow after being broken apart to the level of a single cell. Next, increasing in complexity, the phylum Cnidaria includes jellyfish and coral. Members of this phylum are radially symmetric and diploblastic (possessing two tissue layers). These tissue layers are called the endoderm, which makes up the inner layer, and the ectoderm, which forms the outer layer. Organisms in phylum Cnidaria contain a simple gut with a single opening through which food enters and waste is excreted. Both Porifera and Cnidaria include organisms that are largely sedentary as adults. However, the remaining phyla have evolved adaptations that allow movement and more sophisticated behavior.
Following Cnidaria, there are two phyla of worms, termed Platyhelminthes and Annelida. Worms from both of these phyla breathe by exchanging gases across their skin. As a result, most worms have a large surface area to volume ratio and live in moist or aquatic environments. The skin of these organisms must remain moist to facilitate effective diffusion of gases. Another common characteristic of worms is their bilateral symmetry. This means that an organism can be symmetrically divided in half along a plane. Bilateral symmetry is seen not only in worms, but also in every phyla from this evolutionary point forward. Such symmetry is important to allow directed movement and to concentrate sensory organs near the anterior tip, or head, where they interact with their surroundings. Worms and later phyla also contain an additional tissue layer called the mesoderm that lies between the ectoderm and endoderm. As a result, these organisms are considered &#8220;triploblastic.&#8221;
Of the two phyla of worms, Platyhelminthes is the more ancient and simple. These include flatworms and trematodes, which may be free-living or parasitic. Like Cnidarians, they possess a simple gut with a single opening. Phylum Annelida is more complex, including earthworms, leaches, and polychaeta. These organisms contain a body cavity, called the coelom, between the gut and body wall. They are also distinct due to the segmentation of their bodies. Division of an organism into segments was an important evolutionary step, allowing adaptation of each segment to specialized forms and functions. Major evolutionary advances in this phyla include the development of anterior segments with appendages specialized for grasping and processing food as well as posterior segments with appendages specialized for locomotion.
The next phylum, Mollusca, contains highly morphologically diverse organisms, including octopuses, snails, slugs, and clams. There are few defining features for this group. However, all mollusks possess a mantle, which acts as a protective cover for the respiratory organs, digestive tract, and reproductive structures. Next, the most diverse group of organisms is phylum Arthropoda. This phyla alone makes up approximately three quarters of all living and extinct species known today, including crustaceans, insects, and arachnids. All members of this phylum have a hard, outer coating made of chitin, called an exoskeleton. This exoskeleton is periodically shed for a new, larger one to take its place. Like Annelida, arthropods are segmented. However, instead of multiple repeating sections, arthropods are broken up into three distinct segments called the head, thorax, and abdomen. Most have specialized sensing structures attached to the head called antennae.
Lastly, phylum Chordata includes all animals with a backbone and a few without. Two defining features that unite this group are the presence of a nerve chord and a notochord. The nerve cord is a major part of the chordate nervous system and the notochord is a cartilaginous structure that runs between the nerve cord and the digestive tract. Phylum Chordata includes tunicates (which lose their nerve cord and notochord after metamorphosis from the larval to adult life stage), fish, amphibians, birds, reptiles, and mammals (including humans)3.
<h4>Form and Function</h4>
The vast diversity in body structure, habitats, and behavior observed across all 36 phyla within Kingdom Animalia is truly astounding. To understand these differences better, it is important to consider the connection between form and function. &#8220;Form&#8221; simply means the shape, size, and substance of a structure or organism while &#8220;function&#8221; stands for the way in which that structure is used. Accordingly, the function of a structure can be predicted based on the form observed, or vice versa. For example, a bird specialized to crack hard nuts would be predicted to have a sturdy, sharp beak while a bird that eats fruit may have a smaller, more rounded beak.
Importantly, the form of an organism is constrained by its physiological requirements. For instance, all animals must consume and digest organic matter and exchange gas with their environment. As a result, any adaptations to an animal&#8217;s form must continue to facilitate these processes efficiently. For example, in smaller animals, diffusion of gases across the skin is possible and often useful. However, as the size of an animal increases, its surface area to volume ratio decreases until diffusion across the skin can no longer provide sufficient gas exchange. Such larger animals have developed complex systems to deal with gas exchange, such as the lungs of a whale or the gills of a fish. As a result, the morphology of gas exchange systems depends on both the size of an organism and its environment.
Similarly, it generally follows with other types of adaptations that form follows function, constrained only by the physiological requirement of the organism. However, there are cases in which form does not follow function. Vestigial structures are those that served a purpose in an organism&#8217;s ancestor but are no longer useful in their current form. Such structures have little to no impact on the fitness of an individual, and are thus not evolutionarily selected for or against within the population. As a result, some structures persist with limited functional capacity. For example, the human appendix, a small pocket at the end of the large intestine, is an evolutionary remnant from our ancestors. While this structure has been hypothesized to store beneficial gut bacteria, removal of the appendix is possible with no deleterious effects.
<h4>Convergent Evolution</h4>
Among adaptations seen in Kingdom Animalia, many have developed multiple times throughout evolutionary history. Consider the wings of a bat and those of a bird as an example. These structures are not a result of evolutionary relatedness, but have instead arisen from convergent evolution4. Convergent evolution occurs when two organisms independently evolve similar traits due to similar environmental pressures or niches. The wings of bats, birds, and insets have all developed independently and are said to be &#8220;convergent traits.&#8221; Clearly, such traits are quite beneficial for the niches in which they arise.
<h4>References</h4>
<ol class="references">
	<li>Hollins, J., et al. (2018). 'A physiological perspective on fisheries-induced evolution.' Evol Appl 11(5): 561-576.</li>
	<li>Leong, M., et al. (2017). 'The Habitats Humans Provide: Factors affecting the diversity and composition of arthropods in houses.' Sci Rep 7(1): 15347.</li>
	<li>Wray, G. A. (2015). 'Molecular clocks and the early evolution of metazoan nervous systems.' Philos Trans R Soc Lond B Biol Sci 370(1684).</li>
	<li>Foll, M., et al. (2014). 'Widespread signals of convergent adaptation to high altitude in Asia and America.' Am J Hum Genet 95(4): 394-407.</li>
</ol>

Animal Diversity: Form and Function of Crickets and Crayfish - Concept

동물의 다양성

Kingdom Animalia is composed of a range of organisms united by a set of common characteristics. Barring a few exceptions, animals are multicellular ...

Microbial and Fungal Diversity

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this reason, it is important to understand and distinguish between individual species of these groups. As you will recall, biological taxonomists group organisms based on their phylogenetic relatedness. The three domains of life, Bacteria, Archaea, and Eukaryota segregate life into three distinct groupings. While all bacteria fall within the domain Bacteria, fungi are found in the domain Eukaryota (Kingdom Fungi). In addition to Archaea, domain Bacteria exclusively includes single-celled prokaryotes, meaning their cells lack both a nuclear membrane and membrane-bound organelles. In contrast, fungi of domain Eukaryota have membrane-bound organelles and include both unicellular and multicellular organisms.
<h4>Bacteria</h4>
Bacteria are found nearly everywhere on Earth and are highly adapted to the environments in which they grow. As a result, this domain includes a staggering amount of diversity. The impact of these organisms on human health is equally diverse. For example, species like Streptococcus thermophilus can be used to produce fermented foods like yogurt, while spores of the bacteria Bacillus anthracis are responsible for the potentially deadly disease anthrax.
To aid in the identification of such organism, microbiologist have developed a myriad of tests to characterize both individual cells and entire colonies of bacteria1. One of the most common methods for identifying bacteria is by Gram staining. This test uses colored dyes to determine the composition of the bacteria&#8217;s cell wall. Gram-positive bacteria, which bear a thick cell wall made up of a protein called peptidoglycan, retain a dye called crystal violet and thus appear purple. In contrast, gram-negative bacteria have a thinner cell wall without peptidoglycan and do not retain the crystal violet. Instead, these cells appear pink due to a counter stain called safranin. The use of Gram staining often serves as a preliminary step in the identification of a bacterial species. Further classification requires the identification of additional characteristics. This can include the shape of individual bacteria, which may be either cocci (round), spirillum (spiral), or bacilli (rod-shaped). Additionally, the color, shape, sheen, texture, and even smell of bacterial colonies can be used to distinguish between species.
<h4>Fungi</h4>
Fungi of domain Eukaryota are also highly ecologically diverse and can range in size from microscopic yeast to the largest organism on earth, the honey fungus (Armillaria ostoyae). All fungi are heterotrophic, meaning that they must acquire their food from the environment. These organisms also exhibit diversity in their specific activities, niches, and potential effects on human health. For example, some fungi are consumed or used to produce food by humans while others can cause severe or even deadly infections2.
Within kingdom Fungi, two distinct taxonomic divisions exist based on the reproductive strategy of the species. First, Ascomycota, known as the sac fungi, have a reproductive structure called an ascus. Together, many asci form the ascocarp, or &#8220;fruiting body&#8221; of the fungi. These fungi can reproduce sexually or asexually by budding. Ascomycota consist of some economically important species like the yeast used in brewing beer and baking bread, and some species popular in cuisine, such as morels and truffles. The second group, Basidiomycota, includes organisms commonly known as &#8220;club fungi&#8221; due to the presence of a large club-shaped reproductive structure called the basidium. In contrast to Ascomycota, most Basidomucota reproduce exclusively by sexual reproduction. This occurs when two haploid mycelia join to form a basidiocarp. On a button mushroom, this is the cap structure. On the underside of the basidiocarp, reproductive structures called gills lined with structures called basidia produce haploid nuclei that eventually form basidiospores. Basidiospores are then dispersed by the wind. This taxonomic division includes many commonly eaten button mushrooms, as well as wild puff mushrooms.
<h4>Importance of Microbiology</h4>
Overall, it is clear that the diverse functions and properties of microorganisms make their identification extremely important for human health and well-being. While a vast number of species of fungi and bacteria have been identified to date, it is estimated that millions more have yet to be identified and classified. In addition, human disciplines that explore Fungi and Bacteria are as diverse as the organisms themselves and will undoubtedly continue to grow. The relevance of microorganisms to the medical fields is more prevalent now then ever, as multi-antibiotic resistant species of infectious bacteria threaten human health. Understanding the structure and biological characteristics of infectious and non-infectious species provides key insights necessary for the creation of new antibiotic treatments and preventative strategies3-4.
Microbiology is also critical to the food industry. Numerous products, including yogurt, cheese, kombucha, bread, and alcohol are made using specific categories or species of microbes. At the same time, food scientists focus on engineering chemical compounds and preservatives that will reduce the growth of unwanted microbes on food. In all these processes, an understanding of how microbes survive, reproduce, and affect their environment is necessary.
<h4>References </h4>
<ol class="references">
	<li>Hiroshi Asakura, Holger Brueggemann, Sou-ichi Makino, and Yoshiko Sugita-Konishi, &#8220;Molecular Approaches for the Classification of Microbial Pathogens of Public Health Significance,&#8221; BioMed Research International, vol. 2014, Article ID 725801, 2 pages, 2014. https://doi-org.bdigitaluss.remotexs.co/10.1155/2014/725801.</li>
	<li>Treseder, K. K. and J. T. Lennon (2015). 'Fungal traits that drive ecosystem dynamics on land.' Microbiol Mol Biol Rev 79(2): 243-262.</li>
	<li>Arvanitis, M. and E. Mylonakis (2015). 'Fungal-bacterial interactions and their relevance in health.' Cell Microbiol 17(10): 1442-1446.</li>
	<li>Aarestrup, F. M., et al. (2008). 'Resistance in bacteria of the food chain: epidemiology and control strategies.' Expert Rev Anti Infect Ther 6(5): 733-750.</li>
</ol>

Microbial Colony and Fungal Diversity - Concept

미생물 및 곰팡이 다양성

Bacteria and fungi are two highly diverse groups of organisms that can have significant beneficial or detrimental impacts on human health. For this ...

Cell Division

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Preparation of Onion Root Tips and Solutions
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">For the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water to grow roots for several days. 
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, clean the onion roots of any dirt or debris. 
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Next, in a 1.5 mL tube, dissolve 10 mcg of nocodazole per 1 mL of dimethyl sulfoxide solution, making 1 tube per student. 
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Finally, set out all of the other tools, materials, and reagents from the list of materials onto each student&#39;s lab bench. 
</div></li></ul></li></ol>

Cell Division: Observing Cell Cycle in a Root Tip - Prep

세포 분열

Preparation of Onion Root Tips and Solutions
ExpandFor the cell cycle experiment using root tips, first, leave an onion suspended over a beaker of water ...

세포 과정

Lab Manual Sub Articles

Bacterial Transformation

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Bacterial Transformation
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">First, ensure that each student or student group has a filled ice bucket.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Then, set up a tube rack for each work station.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Into the tube racks, place two empty, sterile 1.5 mL tubes, and then two one mL aliquots of SOC media.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Then, place the plasmid tube into the ice bucket and add two tubes of competent E. coli cells, allowing them to thaw if frozen.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set out two LB agar plates per group and two LB agar ampicillin plates per group.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Ensure the students have the appropriate pipettes.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">And finally, set a water bath to 42 &#176;C and a temperature-controlled shaker to 37 &#176;C.
</div></li></ul></li></ol>

Bacterial Transformation using Plasmids - Prep

박테리아 형질전환

Bacterial Transformation
ExpandFirst, ensure that each student or student group has a filled ice bucket.
Then, set up a tube rack for each work station.
...

Cell Structure

<ol class="note-items"><li data-note-item="1" data-parent-collapse="">Visualizing Onion and Cheek Cells
<button class="expand">Expand</button><ul class="lab-items"><li data-sub-note-item="1-1"><div class="lab-step">Immediately before the experiment, wash and peel onion bulbs for the class.
</div></li><li data-sub-note-item="1-2"><div class="lab-step">Remove the entire brown outer skin and cut the onion in half with a knife. Pull apart the layers of the onion. The thin, nearly transparent film layers within the onion will be used by the students.
</div></li><li data-sub-note-item="1-3"><div class="lab-step">Place the onion film into a Petri dish and then place this into the refrigerator until the students arrive.
</div></li><li data-sub-note-item="1-4"><div class="lab-step">Bring out the microscopes and set them on the lab bench.
</div></li><li data-sub-note-item="1-5"><div class="lab-step">Set the microscopes to the lowest magnification and plug them into an electrical outlet.
</div></li><li data-sub-note-item="1-6"><div class="lab-step">Clean the lenses with lens paper or laboratory wipes and test the knobs to make sure the lenses focus properly.
</div></li><li data-sub-note-item="1-7"><div class="lab-step">Make sure that the stage can be moved.
</div></li></ul></li></ol>

Cell Structure: Visualizing Onion and Human Cells - Prep

세포 구조

Visualizing Onion and Cheek Cells
ExpandImmediately before the experiment, wash and peel onion bulbs for the class.
Remove the entire brown outer skin and ...