Name: retroviral transduction of bone marrow progenitor cells to generate t-cell receptor retrogenic mice
Uploaded: 2016-07-11T15:00:00.000+00:00
Duration: 9 min 8 s
Description: Watch this Scientific Journal Video about Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice at JoVE.com

This work was supported by grants from NIH (5K22A1119151-01 and 1R56DK104903-01) to M.L.B, Pilot/Feasibility Program of the Diabetes Research Center (P30-DK079638) at BCM, JDRF 1-FAC-2014-243-A-N APF, ADA 1-15-JF-07, AAI Careers in Immunology Fellowship to M.B., and The Robert and Janice McNair Foundation.

Ethics Statement: Every effort is made to keep animal discomfort or stress to a minimum during irradiation and tail vein injections. Mice are used as a source of cells in these experiments; as such there are no procedures or manipulations apart from euthanasia. Mice will be euthanized by CO2 inhalation followed by cervical dislocation to confirm death. This procedure is consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.
1. Prepare Retroviral Construct
<ol>
	<li>Sub-clone the T-cell receptor (TCR) alpha and beta chains, separated by a 2A linker, into a MSCV-based retroviral11 vector (example, pMIAII or pMIGII)11. 
	NOTE: The 2A linker allows for stoichiometric and concordant expression of the alpha and beta TCR chains from a single open reading frame. The vector includes an IRES fluorescent protein cassette (ametrine or GFP) that is used to track transduced cells and transduction efficiency. For a detailed protocol please see Holst et al.11</li>
	<li>Isolate Plasmid DNA using commercially obtained plasma prep kit or a similar product. Use a working concentration of 1 - 2 &#956;g &#956;l-1.</li>
</ol>
2. Generation of Retroviral Producer Cell Lines
<ol>
	<li>Utilize a two-step process to produce the retroviral producer cell lines. 
	NOTE: For a detailed protocol, please view Holst et al.11.
	<ol>
		<li>Generate retrovirus by transient transfection of 293T cells with three plasmids (pVSVG, pEQ-Pam3(-E) and vector of interest11) and take the retroviral supernatant from the 293T cells to transduce the GP+E86 ecotropic retroviral producer cell line.</li>
		<li>Isolate the top 40% of fluorescent expressing GP+E86 producer cells by FACS and propagate the transdcuced-GP+E86 producer cells as a source of virus. 
		NOTE: The generation of high-titer producers is critical for efficient transduction of bone marrow cells. For a complete protocol please view Holst et al.11</li>
	</ol>
	</li>
</ol>
3. Retrovirus-mediated Stem Cell Gene Transfer (Day -5)&#160;
<ol>
	<li>Thaw out and culture 0.5 -1.0 x 106 of each GP+E86 producer cell line in complete DMEM 10% (vol/vol) FBS to allow sufficient growth for two confluent 150-mm plates by Day 0 of this protocol. 
	NOTE: Thawing 0.5 - 1.0 x 106 of each GP+E86 producer cell line in a 10 cm plate will allow for 30 - 50% confluency at Day 5. This also allows for 5 days of culture in order to expand the GP+E86 producer cell lines to two confluent 150-mm plates by Day 0.
	<ol>
		<li>Culture retroviral producer cell line in complete tissue culture media containing 5% feta calf serum, and ciprofloxacin (10 &#956;g/ml) to avoid mycoplasma contamination. 
		NOTE: Discontinue use of ciprofloxacin when generating the viral supernatant for bone marrow transduction. Avoid over-growth of the producer cell line, as this will negatively impact bone marrow transduction efficiency.</li>
	</ol>
	</li>
</ol>
4. Inject Donor Mice with 5-Fluorouracil (5-FU) (Day -3)
<ol>
	<li>Weigh the donor mice (5 - 12 weeks old) to determine the amount of 5-FU required. Using mice younger than 5 weeks of age results in low bone marrow yield. To ensure expression of a single TCR, use a T cell deficient mouse strain such as SCID, RAG or TCR alpha deficient mice as bone marrow donors.</li>
	<li>Working in the tissue culture hood, dilute the 5-FU stock with sterile PBS in order to make a sufficient amount of 10 mg ml-1 working solution of 5-FU to deliver 0.15 mg 5-FU per gram of body weight.</li>
	<li>Using a 1 ml syringe with a 37 G needle, intraperitoneal inject 0.15 mg 5-FU per gram of body weight per donor mouse. 
	NOTE: Use 1 donor mouse per one recipient to start and adjust the number of donor mice according to the cell yield. An alternative method to the 5-FU treatment for the enrichment and isolation of bone marrow progenitors is negative selection of lineage positive cells as described in Viret et al.</li>
</ol>
5. Bone Marrow Extraction Procedure (Day 0)
<ol>
	<li>Obtain dissection scissors and forceps for bone isolation. Prepare media (HBSS supplemented with 5% FCS (vol/vol)) to collect bones in a 50 ml conical tube. Keep 50 ml conical containing media on ice. Euthanize mice by CO2 inhalation followed by cervical dislocation to confirm death. Pinch paw to ensure no reflexes are detected.</li>
	<li>Sterilize the surgical site and surgical tools with ethanol or methanol. Remove the femur, tibia, humerus, and pelvic girdle carefully by first cutting up and under the pelvic girdle. Cut back down along the femur to the tibia. Remove all surrounding tissue using scissors and forceps to obtain clean bones. Keep bones hydrated in HBSS + 5% (vol/vol) FBS.</li>
	<li>Separate the bones by cutting at the joints, making sure to cut both ends off each bone. Insert a 25-gauge needle attached to a 10-ml syringe containing HBSS + 5% (vol/vol) FBS. Apply pressure and flush all bone marrow through a 70-&#956;m strainer resting in a 50 ml conical.</li>
	<li>Periodically, push the bone marrow through the 70-&#956;m filter using the plunger from a 1 ml or 3 ml syringe. Rinse the strainer with HBSS + 5% (vol/vol) FBS. This will ensure a single cell suspension that is free of bone fragments and tissue. Keep cells sterile by performing the procedure in a sterile hood.</li>
	<li>Centrifuge bone marrow cells at 300 x g for 10 min at 4 &#176;C.</li>
</ol>
6. Bone Marrow Culture (Day 0)
<ol>
	<li>Decant or aspirate media and resuspend cell pellet in 1 ml ammonium chloride based red blood lysis buffer (or RBC lysis buffer equivalent) per 50 ml conical tube. After 1 min incubation at room temperature, quench the red cell lysis buffer by adding 10 ml of complete DMEM 20% (vol/vol) FBS.</li>
	<li>Centrifuge cells at 300 x g for 10 min at 4 &#176;C.</li>
	<li>Decant or aspirate supernatant and resuspend bone marrow in 5 ml complete DMEM 20% (vol/vol) FBS. Count the cells with a Neubauer counting chamber. 
	NOTE: General yield should be approximately 5 - 10 x 106 cells per mouse; however, the yield can vary among different strains of mice.</li>
	<li>Plate bone marrow cells at a density of 4 x 107 cells per 150-mm plate in 30 ml of complete DMEM 20% (vol/vol) FBS supplemented with IL-3 (20 g ml-1), IL-6 (50 g ml-1) and SCF (50 g ml-1). 
	NOTE: Use freshly thawed-aliquoted cytokines. Do not use cytokines that have been frozen and thawed more than once or kept at 4 &#176;C for more than 2 weeks.</li>
	<li>Incubate bone marrow overnight (approximately 24 hr) at 37 &#176;C with 5% CO2.</li>
</ol>
7. Retroviral Producer Cell Culture (Day 0)
<ol>
	<li>Plate 3 x 106 retroviral producer cells per 150-mm plate in 18 ml complete DMEM 20% (vol/vol) FBS.</li>
	<li>Incubate retroviral producer cells at 37 &#176;C overnight. Anticipate one 150-mm plate for 15 million bone marrow cells (approximately 2 - 3 donor mice).</li>
</ol>
8. Retroviral Supernatant (Day 1)
<ol>
	<li>The next day (approximately 24 hr later), harvest the retroviral supernatant from the producer plates (set up in step 7) by drawing up the retroviral supernatant directly off of the 150-mm plate with a 10-ml syringe and filter using a 0.45-&#956;m syringe filter.</li>
	<li>Carefully replace the removed retroviral supernatant with 18 ml of fresh complete DMEM 20% (vol/vol) FBS.</li>
	<li>Supplement the filtered retroviral supernatant with IL-3 (20 g ml-1), IL-6 (50 g ml-1), MSCF (50 g ml-1) and Hexadimethrine bromide (Polybrene) (6 &#956;g ml-1). 
	NOTE: Hexadimethrine bromide should be made fresh every 2 months to avoid a drop in efficacy of retroviral transduction.</li>
</ol>
9. Bone Marrow Harvest (Day 1)
<ol>
	<li>After completing step 8.3, harvest the bone marrow cells from Step 6.4 and transfer the media containing non-adherent cells into sterile 50-ml tubes.</li>
	<li>Wash the plates vigorously with 10 ml PBS and collect in the 50 ml conical from step 9.1. If necessary, repeat the wash step.</li>
	<li>Centrifuge the cells at 300 x g for 10 min at 4 &#176;C, decant the supernatant, resuspend cells in a small volume (1 - 3 ml) complete DMEM 20% (vol/vol) FBS, and count. Anticipate 50 - 75% recovery from day 0. Resuspend bone marrow at 1 x 106 cells per 1 ml of the retroviral supernatant containing cytokines and Hexadimethrine bromide.</li>
</ol>
10. Bone Marrow Transduction (Day 1)
<ol>
	<li>Plate the retroviral supernatant/bone marrow mixture at a volume of 3 ml per well in a six-well tissue culture plate. Wrap the plates in plastic wrap to minimize gas exchange during the centrifugation in step 10.2.</li>
	<li>Spin the wrapped six-well plates at 1,000 x g for 60 min a 37 &#176;C.</li>
	<li>After the spin is complete, remove the plastic wrap and place the plates in a 37 &#176;C CO2 incubator for 24 hr.</li>
</ol>
11. Retroviral Supernatant and Transduction (Day 2)
<ol>
	<li>After 24 hr, collect fresh viral supernatant from the viral producer cell line. Filter the retroviral supernatant using a 10-ml syringe and a 0.45-&#956;m syringe filter. Supplement the filtered retroviral supernatant with IL-3 (20 g ml-1), IL-6 (50 g ml-1) and SCF (50 g ml-1) and Hexadimethrine bromide (6 g ml-1).</li>
	<li>Next, carefully remove with pipette or aspirate the top 2 ml of media from each well of the six-well plate containing the bone marrow cells. 
	NOTE: Work carefully not to aspirate the bone marrow, which is usually concentrated in the middle of the well. Alternatively, the removed supernatant can be spun down to recover any lost bone marrow cells.</li>
	<li>To each well in the bone marrow 6-well plate add 3 ml of fresh viral supernatant containing cytokines and Hexadimethrine bromide. Wrap the plates in plastic, and repeat the spin transduction at 1,000 x g for 60 min a 37 &#176;C. Unwrap the plates, and incubate cells at 37 &#176;C and 5% CO2 overnight (about 24 hr).</li>
</ol>
12. Addition of Fresh Supplemented DMEM (Day 3)
<ol>
	<li>After 24 hr, remove the top 2 ml of media from each well of the six-well plate containing the bone marrow cells, as in step 11.2. Replace the removed media with fresh DMEM 20% (vol/vol) FBS supplemented with final concentrations of IL-3 (20 g ml-1), IL-6 (50 g ml-1) and SCF (50 g ml-1).</li>
	<li>Incubate bone marrow plates at 37 &#176;C CO2 incubator for another 24 hr.</li>
</ol>
13. Harvest Transduced Bone Marrow (Day 4)
<ol>
	<li>After 24 hr, collect the bone marrow cells from the six-well plates. Wash the plates vigorously with PBS to remove all non-adherent cells.
	<ol>
		<li>Centrifuge the cells at 300 x g for 10 min at 4 &#176;C and decant the supernatant.</li>
		<li>Resuspend the bone marrow in a small volume (1 ml) of PBS + 0.5% (vol/vol) heat-inactivated FBS. Keep cells on ice.</li>
	</ol>
	</li>
	<li>Take a 10-&#956;l sample of each bone marrow condition and count the cells using a Neubauer counting chamber. Assuming 1 - 2 donor per recipient, the yield will be sufficient to transfer at least 4 x 106 cells per recipient mouse. Resuspend the bone marrow in a sufficient volume of PBS + 0.5% (vol/vol) heat-inactivated FBS to inject 200 - 300 &#956;l per mouse.
	<ol>
		<li>Inject at least 2 x 106 cells with a transduction efficiency of 25% (can inject up to 8 x106 cells) per mouse intravenously. 
		NOTE: We routinely inject five recipient mice per two 6-well plates of bone marrow cells. If the yield is substantially lower, more donor mice should be used until the sufficient bone marrow number can be obtained. In order to achieve optimal reconstitution, at least 5 x 105 transduced (fluorescent positive) should be injected into each recipient.</li>
	</ol>
	</li>
	<li>With a micro pipettor, take a 10-&#956;l sample of each bone marrow condition and analyze transduction efficiency by flow cytometry based on expression of the fluorescent marker (Figure 1A)11. 
	NOTE: Generally, the percentage of fluorescent positive cells is between 25% and 70%, depending on the construct and retroviral titer. The number of transduced bone marrow cells necessary to reconstitute a sub-lethally irradiate mouse can vary depending on the transduction efficiency. The lower the transduction efficiency, the more bone marrow cells required and conversely, the higher the bone marrow transduction efficiency, the lower number of cells required for reconstitution. Transduction efficiency below 25% is suboptimal and may result in insufficient reconstitution and survival. In order to ensure optimal bone marrow recovery and transduction efficiency, use freshly prepared tissue culture media and cytokines. When expressing a new TCR utilizing the retrogenic system, the selection of that TCR in vivo is unknown. Depending on each individual TCR affinity, TCR selection in the thymus and peripheral T cell expression will vary17.</li>
</ol>
14. Mouse Irradiation, Bone Marrow Injection and Care
<ol>
	<li>Irradiate mice the day before bone marrow injection (Same day as Step 13). Use the following doses: C57BL/6 mice (and other wild type strains), 900 rads; Rag1-/- mice, 500 rads; scid mice, 300 rads).
	<ol>
		<li>Place recipient mice on Amoxicillin (50 mg/kg/day) or another antibiotic water treatment before irradiation, and keep on antibiotics for the first two weeks after bone marrow injection. 
		NOTE: Optimal irradiation dose may have to be determined empirically.</li>
	</ol>
	</li>
	<li>For injection, load the cells from Step 13.1 into a 1-ml syringe using blunt needles immediately before injection, then change the needle to 27-gauge for injection, expel any air to avoid air embolism. Heat mice under a heating lamp and inject 0.2 ml of bone marrow per mouse intravenously. 
	NOTE: Do not let cells sit in the syringe for any length of time or the cells will settle.</li>
	<li>Monitor the mice weekly to ensure that they remain healthy.</li>
	<li>After 5 - 6 weeks, bleed the mice to check for reconstitution on the basis of GFP+/ametrine+ and TCR co-expression (Figure 1B)11.</li>
</ol>

<ol>
	<li>Qi, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+and+clonal+selection+in+the+human+T-cell+repertoire.">Diversity and clonal selection in the human T-cell repertoire.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, 13139-13144 (2014).</li><li>Zarnitsyna, V. I., Evavold, B. D., Schoettle, L. N., Blattman, J. N., Antia, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+diversity,+completeness,+and+cross-reactivity+of+the+T+cell+repertoire.">Estimating the diversity, completeness, and cross-reactivity of the T cell repertoire.</a> Frontiers in immunology. 4, 485 (2013).</li><li>Kisielow, P., Bluthmann, H., Staerz, U. D., Steinmetz, M., von Boehmer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+in+T-cell-receptor+transgenic+mice+involves+deletion+of+nonmature+CD4+8++thymocytes.">Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes.</a> Nature. 333, 742-746 (1988).</li><li>Hogquist, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cell+receptor+antagonist+peptides+induce+positive+selection.">T cell receptor antagonist peptides induce positive selection.</a> Cell. 76, 17-27 (1994).</li><li>Verdaguer, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+autoimmune+diabetes+in+monoclonal+T+cell+nonobese+diabetic+mice.">Spontaneous autoimmune diabetes in monoclonal T cell nonobese diabetic mice.</a> J Exp Med. 186, 1663-1676 (1997).</li><li>Katz, J. D., Wang, B., Haskins, K., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Following+a+diabetogenic+T+cell+from+genesis+through+pathogenesis.">Following a diabetogenic T cell from genesis through pathogenesis.</a> Cell. 74, 1089-1100 (1993).</li><li>Pauza, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-cell+receptor+transgenic+response+to+an+endogenous+polymorphic+autoantigen+determines+susceptibility+to+diabetes.">T-cell receptor transgenic response to an endogenous polymorphic autoantigen determines susceptibility to diabetes.</a> Diabetes. 53, 978-988 (2004).</li><li>Jasinski, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+insulin+(B:9-23)+T-cell+receptor+mice+develop+autoimmune+diabetes+dependent+upon+RAG+genotype,+H-2g7+homozygosity,+and+insulin+2+gene+knockout.">Transgenic insulin (B:9-23) T-cell receptor mice develop autoimmune diabetes dependent upon RAG genotype, H-2g7 homozygosity, and insulin 2 gene knockout.</a> Diabetes. 55, 1978-1984 (2006).</li><li>Kersh, G. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR+transgenic+mice+in+which+usage+of+transgenic+alpha-+and+beta-chains+is+highly+dependent+on+the+level+of+selecting+ligand.">TCR transgenic mice in which usage of transgenic alpha- and beta-chains is highly dependent on the level of selecting ligand.</a> Journal of immunology. 161, 585-593 (1998).</li><li>Bettini, M. L., Bettini, M., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR+retrogenic+mice:+A+rapid,+flexible+alternative+to+TCR+transgenic+mice.">TCR retrogenic mice: A rapid, flexible alternative to TCR transgenic mice.</a> Immunology. 136 (3), 265-272 (2012).</li><li>Holst, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T-cell+receptor+retrogenic+mice.">Generation of T-cell receptor retrogenic mice.</a> Nat Protoc. 1, 406-417 (2006).</li><li>Holst, J., Vignali, K. M., Burton, A. R., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+analysis+of+T-cell+selection+in+vivo+using+T+cell-receptor+retrogenic+mice.">Rapid analysis of T-cell selection in vivo using T cell-receptor retrogenic mice.</a> Nat Methods. 3, 191-197 (2006).</li><li>Bettini, M. L., Bettini, M., Nakayama, M., Guy, C. S., Vignali, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+T+cell+receptor-retrogenic+mice:+improved+retroviral-mediated+stem+cell+gene+transfer.">Generation of T cell receptor-retrogenic mice: improved retroviral-mediated stem cell gene transfer.</a> Nat Protoc. 8, 1837-1840 (2013).</li><li>Donnelly, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+aphthovirus+2A/2B+polyprotein+'cleavage'+mechanism+indicates+not+a+proteolytic+reaction,+but+a+novel+translational+effect:+a+putative+ribosomal+'skip'.">Analysis of the aphthovirus 2A/2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'.</a> J Gen Virol. 82, 1013-1025 (2001).</li><li>Atkins, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+for+"StopGo":+reprogramming+translation+to+augment+codon+meaning+of+GGN+by+promoting+unconventional+termination+(Stop)+after+addition+of+glycine+and+then+allowing+continued+translation+(Go).">A case for "StopGo": reprogramming translation to augment codon meaning of GGN by promoting unconventional termination (Stop) after addition of glycine and then allowing continued translation (Go).</a> RNA. 13, 803-810 (2007).</li><li>Doronina, V. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+release+of+nascent+chains+from+ribosomes+at+a+sense+codon.">Site-specific release of nascent chains from ribosomes at a sense codon.</a> Mol Cell Biol. 28, 4227-4239 (2008).</li><li>Bettini, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TCR+affinity+and+tolerance+mechanisms+converge+to+shape+T+cell+diabetogenic+potential.">TCR affinity and tolerance mechanisms converge to shape T cell diabetogenic potential.</a> Journal of immunology. 193, 571-579 (2014).</li><li>Brehm, M. A., Wiles, M. V., Greiner, D. L., Shultz, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+improved+humanized+mouse+models+for+human+infectious+diseases.">Generation of improved humanized mouse models for human infectious diseases.</a> J Immunol Methods. 410, 3-17 (2014).</li><li>Brehm, M. A., Shultz, L. D., Greiner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Humanized+mouse+models+to+study+human+diseases.">Humanized mouse models to study human diseases.</a> Curr Opin Endocrinol Diabetes Obes. 17, 120-125 (2010).</li><li>Chaplin, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+interleukin-12+as+a+self-processing+2A+polypeptide.">Production of interleukin-12 as a self-processing 2A polypeptide.</a> J Interferon Cytokine Res. 19, 235-241 (1999).</li><li>Collison, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inhibitory+cytokine+IL-35+contributes+to+regulatory+T-cell+function.">The inhibitory cytokine IL-35 contributes to regulatory T-cell function.</a> Nature. 450, 566-569 (2007).</li><li>Holst, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scalable+signaling+mediated+by+T+cell+antigen+receptor-CD3+ITAMs+ensures+effective+negative+selection+and+prevents+autoimmunity.">Scalable signaling mediated by T cell antigen receptor-CD3 ITAMs ensures effective negative selection and prevents autoimmunity.</a> Nature immunology. 9, 658-666 (2008).</li><li>Kalos, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+cells+with+chimeric+antigen+receptors+have+potent+antitumor+effects+and+can+establish+memory+in+patients+with+advanced+leukemia.">T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.</a> Sci Transl Med. 3, 95ra73 (2011).</li><li>VanSeggelen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T+Cells+Engineered+With+Chimeric+Antigen+Receptors+Targeting+NKG2D+Ligands+Display+Lethal+Toxicity+in+Mice.">T Cells Engineered With Chimeric Antigen Receptors Targeting NKG2D Ligands Display Lethal Toxicity in Mice.</a> Mol Ther. 23, 1600-1610 (2015).</li></ol>

No conflicts of interest declared.

In the protocol, we detail several critical steps to ensure optimal bone marrow health, transduction efficiency and reconstitution. First critical step is the generation and proper maintenance of the GP+E86 viral producer cells. Use early passage producer cell lines and maintain at 80% confluency or lower prior to use. When making fresh GP+E86 viral producer cells, ensure the 293T cells are early passage and growing in culture for 24 - 48 hr. Plating too many GP+E86 cells during the transduction step will lower viral tit...

T cell receptor (TCR) repertoire of humans and mice has been estimated at 1 x 108 and 2 x 106 unique TCRs respectively1,2. This large diversity allows T cells to recognize a vast array of antigen epitopes derived from self-peptides as well as from pathogens presented by the major histocompatibility complex (MHC) on antigen presenting cells (APCs). The subtle differences in the interactions of the TCRs with unique peptide-MHC complexes dictate whether a T cell will undergo apoptosis, anergy, activation, differentiation, cytokine production or cytotoxicity. However, due to the large TCR repertoire, analysis of how a specific TCR will respond to a particular antigen requires the use of single TCR systems.
Various TCR transgenic mice have been generated in order to study the function of a single TCR in an in vivo model 3-9. However, there are caveats to TCR transgenic mice including the cost, the length of time to generate a single transgenic mouse and the so called founder effect of random transgene insertion into germline DNA10. Therefore, relatively few TCR transgenic mice have been generated for any given antigen and the functional implications of high and low TCR affinity for the same epitope are rarely addressed. To address the need for a rapid approach to screen and study multiple TCRs individually or in combination, retrogenic (&#39;retro&#39; from retrovirus and &#39;genic&#39; from transgenic) mice have been utilized as an alternative to TCR transgenic mice11-13.
The 2A peptide consensus motif found within several viruses consist of an 2A-Asp-Val/Ile-Glut-X-Asn-Pro-Gly-2B-Pro, in which cleavage occurs between the glycine of the 2A and the proline of the 2B from cis-acting hydrolase activity, resulting in ribosomal skipping during translation10,14-16. For a detailed diagram depicting the cleavage of the various 2A peptides (F2A, E2A, T2A and P2A) please refer to references 10 - 12. In this manner, 2 cistrons (TCR alpha and TCR beta) can be linked resulting in stoichiometric translation in a single vector. Utilizing this approach, we are able to express and directly compare multiple antigen specific TCRs in vivo.

<table><tbody><tr><td>DMEM, high glucose + glutamine</td><td>Corning Cellgro</td><td>10-013-CV</td><td>Dulbecco&#039;s Modification of Eagle&#039;s Medium with 4.5 g/L glucose, L-glutamine &amp;amp; sodium pyruvate</td></tr><tr><td>FBS</td><td>Atlanta Biological</td><td>S11550</td><td></td></tr><tr><td>Trypsin-Versene</td><td>Lonza</td><td>17-161F</td><td></td></tr><tr><td>0.45 &amp;#181;m syringe filter</td><td>Thermo Scientific</td><td>194-2545</td><td></td></tr><tr><td>polybrene</td><td>Sigma</td><td>H9268-10G</td><td>Sterile Filtered in dH&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Ciprofloxacin&amp;nbsp;</td><td>VWR</td><td>AAJ61970-06</td><td></td></tr><tr><td>5-fluorouracil (5-FU)</td><td>VWR</td><td>AAA13456-06</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Corning Cellgro</td><td>25-000-CI</td><td></td></tr><tr><td>MEM nonessential Amino Acids</td><td>Corning Cellgro</td><td>25-025-CI</td><td></td></tr><tr><td>HEPES 1 M solution</td><td>Corning Cellgro</td><td>25-060-CI</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Gibco by Life Technologies</td><td>21985-023</td><td></td></tr><tr><td>Pen/Strept</td><td>Corning Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>L-glutamine</td><td>Corning Cellgro</td><td>25-005-CI</td><td></td></tr><tr><td>150 mm tissue culture dishes</td><td>Greiner Bio-one</td><td>639160</td><td></td></tr><tr><td>Tisue culture-treated 6-well flat plate</td><td>Greiner Bio-one</td><td>657160</td><td></td></tr><tr><td>70 &amp;#181;m nylon cell strainers</td><td>Falcon</td><td>352350</td><td></td></tr><tr><td>Mouse IL-3</td><td>Invitrogen</td><td>PMC0033</td><td></td></tr><tr><td>Human IL-6</td><td>Invitrogen</td><td>&amp;nbsp;PHC0063</td><td></td></tr><tr><td>Mouse Stem Cell Factor</td><td>Invitrogen</td><td>PMC2113L</td><td></td></tr><tr><td>10x PBS</td><td>Corning Cellgro</td><td>46-D13-CM</td><td></td></tr><tr><td>HANKS Buffer</td><td>Corning Cellgro</td><td>21020147</td><td></td></tr><tr><td>BD 10 ml Syringe</td><td>BD</td><td>300912</td><td></td></tr><tr><td>BD 1 ml Syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>27 G x 1/2 BD Precision Glide Needle</td><td>BD</td><td>305109</td><td></td></tr><tr><td>30 G x 1/2 BD Precision Glide Needle</td><td>BD</td><td>305106</td><td></td></tr></tbody></table>

retroviral transduction of bone marrow progenitor cells to generate t-cell receptor retrogenic mice

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively. The vast array of TCRs proves studying a specific antigenic response difficult. Therefore, TCR transgenic mice were made to study positive and negative selection in the thymus as well as peripheral T cell activation, proliferation and tolerance. However, relatively few TCR transgenic mice have been generated specific to any given antigen. Thus, studies involving TCRs of varying affinities for the same antigenic peptide have been lacking. The generation of a new TCR transgenic line can take six or more months. Additionally, any specific backcrosses can take an additional six months. In order to allow faster generation and screening of multiple TCRs, a protocol for retroviral transduction of bone marrow was established with stoichiometric expression of the TCR&#945; and TCR&#946; chains and the generation of retrogenic mice. Each retrogenic mouse is essentially a founder, virtually negating a founder effect, while the length of time to generate a TCR retrogenic is cut from six months to approximately six weeks. Here we present a rapid and flexible alternative to TCR transgenic mice that can be expressed on any chosen background with any particular TCR.

Bone marrow transduction efficiency is checked at step 13.3 of the protocol before the bone marrow is injected into tail vein i.v. In the representative bone marrow transduction figure (Figure 1A), approximately 10-&#956;l of the harvested bone marrow was added to 100-&#956;l of PBS and analyzed for ametrine expression. Generally the percentage of fluorescent positive cells is between 25% and 70%, depending on the construct and retroviral titer. After 6 weeks bone marrow ...

Watch this Scientific Journal Video about Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice at JoVE.com

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

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Research

JoVE Journal

Immunology and Infection

9.5K Views.  Baylor College of Medicine. We present a rapid and flexible protocol for a single T cell receptor (TCR) retroviral-based in vivo expression system. Retroviral vectors are used to transduce bone marrow progenitor cells to study T cell development and function of a single TCR in vivo as an alternative to TCR transgenic mice.

Video: Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the gene of interest in hematopoietic cells and can be used to study the gene's role in normal and/or malignant hematopoiesis. This protocol provides a detailed description on how to perform transduction-transplantation using calreticulin (CALR) mutations recently identified in myeloproliferative neoplasm (MPN) as an example. In this protocol whole bone marrow cells from 5-flurouracil (5-FU) treated donor mice are transduced with a retrovirus encoding mutant CALR and transplanted into lethally irradiated syngeneic hosts. Donor cells expressing mutant CALR are marked with green fluorescent protein (GFP). Transplanted mice develop an MPN phenotype including elevated platelets in the peripheral blood, expansion of megakaryocytes in the bone marrow, and bone marrow fibrosis. We provide a step-by-step account of how to generate retrovirus, calculate viral titer, transduce whole bone marrow cells, and transplant into irradiated recipient mice.

This manuscript provides a description of the methodology used to establish transduction-transplantation mouse models. A detailed account is given of technical errors to avoid when performing bone marrow transplants. A clear understanding should be gained of the importance of high viral titer, transfection/transduction, and irradiation.

Transduction-transplantation is a quick and efficient way to model human hematologic malignancies in mice. This technique results in expression of the ...

Cancer Research

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged antigen-specific T cell receptor (TCR) have been an indispensable tool to study T cell development and function. However, such TCRs are usually isolated from the relevant T cells after long-term culture often following repeated antigen stimulation, which unavoidably selects for T cells with high affinity. Random genomic integration of the TCR &alpha;- and &beta;-chain and expression from non-endogenous promoters can lead to variations in expression level and kinetics.
Epigenetic reprogramming via somatic cell nuclear transfer provides a tool to generate embryonic stem cells and mice from any cell of interest. Consequently, when SCNT is applied to T cells of known specificity, these genetic V(D)J rearrangements are transferred to the SCNT-embryonic stem cells (ESCs) and the mice derived from them, while epigenetic marks are reset. We have demonstrated that T cells with pre-defined specificities against Toxoplasma gondii can be used to generate mouse models that express the specific TCR from their endogenous loci, without experimentally introduced genetic modification. The relative ease and speed with which such transnuclear models can be obtained holds promise for the construction of other disease models.

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

We demonstrate here that epigenetic reprogramming via Somatic Cell Nuclear Transfer (SCNT) can be used as a tool to generate mouse models with pre-defined T cell receptor (TCR) specificities. These transnuclear mice express the corresponding TCR from their endogenous locus under the control of the endogenous promoter.

Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

Retroviral Transduction of T-cell Receptors in Mouse T-cells

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen presenting cells (APCs)1. This recognition leads to the activation of T-cells and a series of functional outcomes (e.g. cytokine production, killing of the target cells). Understanding the functional role of TCRs is critical to harness the power of the immune system to treat a variety of immunology related diseases (e.g. cancer or autoimmunity).
It is convenient to study TCRs in mouse models, which can be accomplished in several ways. Making TCR transgenic mouse models is costly and time-consuming and currently there are only a limited number of them available2-4. Alternatively, mice with antigen-specific T-cells can be generated by bone marrow chimera. This method also takes several weeks and requires expertise5. Retroviral transduction of TCRs into in vitro activated mouse T-cells is a quick and relatively easy method to obtain T-cells of desired peptide-MHC specificity. Antigen-specific T-cells can be generated in one week and used in any downstream applications. Studying transduced T-cells also has direct application to human immunotherapy, as adoptive transfer of human T-cells transduced with antigen-specific TCRs is an emerging strategy for cancer treatment6.
Here we present a protocol to retrovirally transduce TCRs into in vitro activated mouse T-cells. Both human and mouse TCR genes can be used. Retroviruses carrying specific TCR genes are generated and used to infect mouse T-cells activated with anti-CD3 and anti-CD28 antibodies. After in vitro expansion, transduced T-cells are analyzed by flow cytometry.

We present a protocol to produce antigen-specific mouse T-cells using retroviral transduction

T-cell receptors (TCRs) play a central role in the immune system. TCRs on T-cell surfaces can specifically recognize peptide antigens presented by antigen ...

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, Bcr-PDGFR&#945;, etc.). We are able to model and study a human-like disease in our mouse model, by transplanting bone marrow cells previously infected with a retrovirus expressing the oncogene of interest. Replication-defective retrovirus encoding a human oncogene and a marker (GFP, RFP, antibiotic resistance gene, etc.) is produced by a transient transfection protocol using 293T cells, a human renal epithelial cell line transformed by the adenovirus E1A gene product. 293 cells have the unusual property of being highly transfectable by calcium phosphate (CaPO4), with up to 50-80% transfection efficiency readily attainable. Here, we co-transfect 293 cells with a retroviral vector expressing the oncogene of interest and a plasmid that expresses the gag-pol-env packaging functions, such as the single-genome packaging constructs kat or pCL, in this case the EcoPak plasmid. The initial transfection is further improved by use of chloroquine. Stocks of ecotropic virus, collected as culture supernatant 48 hrs. post-transfection, can be stored at -80&deg;C and used for infection of cell-lines in view of transformation and in vitro studies, or primary cells such as mouse bone marrow cells, that can then be used for transplant in our mouse model.

This technique demonstrates an efficient way to prepare replication-defective retroviral stocks encoding a human oncogene, and subsequently used for induction of myeloproliferative disease in the mouse model.

Our lab studies human myeloproliferative diseases induced by such oncogenes as Bcr-Abl or growth factor receptor-derived oncogenes (ZNF198-FGFR1, ...

Biology

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, cloning TCRs specific for various tumor-associated antigens has been the goal of many studies. To elicit an effective T cell response, the TCR must recognize the target antigen with optimal affinity. However, cloning such TCRs has been a challenge and many available TCRs possess sub-optimal affinity for the cognate antigen. In this protocol, we describe a method of cloning de novo high affinity antigen-specific TCRs using existing TCRs by exploiting hemichain centricity. It is known that for some TCRs, each TCR&#945; or TCR&#946; hemichain do not contribute equally to antigen recognition, and the dominant hemichain is referred to as the centric hemichain. We have shown that by pairing the centric hemichain with counter-chains differing from the original counter-chain, we are able to maintain the antigen specificity, while modulating its interaction strength for the cognate antigen. Thus, the therapeutic potential of a given TCR can be improved by optimizing the pairing between the centric and counter hemichains.

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Herein we describe a novel method to generate antigen-specific T cell receptors (TCRs) by pairing the TCR&#945; or TCR&#946; of an existing TCR, possessing the antigen-specificity of interest, with complementary hemichain of the peripheral T cell receptor repertoire. The de novo generated TCRs retain antigen-specificity with varying affinity.

T cell receptors (TCRs) are used clinically to direct the specificity of T cells to target tumors as a promising modality of immunotherapy. Therefore, ...

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have been conducive to downstream in vivo functional analysis of TCR heterodimers. We have developed an improved protocol based on a two-step multiplex-nested PCR, which results in a PCR product that spans entire variable regions of a human TCR alpha and beta chains. By identifying unique restriction sites and incorporating them into the PCR primers, we have made the PCR product compatible with direct sub-cloning into the template retroviral vector. The resulting retroviral construct encodes a chimeric human/mouse TCR with a mouse intracellular domain, which is functional in mouse cells or in in vivo mouse models. Overall, the protocol described here combines human single cell paired TCR alpha and beta chain identification with streamlined generation of retroviral vectors readily adaptable for in vitro and in vivo TCR expression. The video and the accompanying material are designed to give a highly detailed description of the single cell PCR, so that the critical steps can be followed and potential pitfalls avoided. Additionally, we provide a detailed description of the cloning steps necessary to generate the expression vector. Once mastered, the whole procedure from single cell sorting to TCR expression could be performed in a short two-week period.

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

The current protocol combines single cell paired human TCR alpha and beta chain sequencing with streamlined generation of retroviral vectors compatible with in vitro and in vivo TCR expression.

Although, several methods for sequencing of paired T cell receptor (TCR) alpha and beta chains from single T cells have been developed, none so far have ...

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting ...

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide further insight on cell function. B-1a cells are a unique B cell subset in mice that produce protective natural IgM antibodies against oxidation-specific epitopes that arise during health and disease. B-1a cell IgM production differs depending on B-1a cell location, and therefore it becomes useful from a therapeutic standpoint to target B-1a localization to niches supportive of high antibody production. Here we describe a method to target B-1a cell migration to the bone marrow by retroviral-mediated overexpression of the C-X-C motif chemokine receptor 4 (CXCR4). Gene induction in primary murine B cells can be challenging and typically yields low transfection efficiencies of 10-20% depending on technique. Here we demonstrate that retroviral transduction of primary murine B-1a cells results in 30-40% transduction efficiency. This method utilizes adoptive cell transfer of transduced B-1a cells into B cell-deficient recipient mice so that donor B-1a cell migration and localization can be visualized. This protocol can be modified for other retroviral constructs and can be used in diverse functional assays post-adoptive transfer, including analysis of donor cell or host cell phenotype and function, or analysis of soluble factors secreted post B-1a cell transfer. The use of distinct donor and recipient mice differentiated by CD45.1 and CD45.2 allotype and the presence of a GFP reporter within the retroviral plasmid could also enable detection of donor cells in other, immune-sufficient mouse models containing endogenous B cell populations.

Here we describe a method for retroviral overexpression and adoptive transfer of murine B-1a cells to examine in vivo B-1a cell migration and localization. This protocol can be extended for diverse downstream functional assays including quantification of donor B-1a cell localization or analysis of donor cell-derived secreted factors post-adoptive transfer.

As cell function is influenced by niche-specific factors in the cellular microenvironment, methods to dissect cell localization and migration can provide ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Retroviral Transduction of Bone Marrow Progenitor Cells to Generate T-cell Receptor Retrogenic Mice

Summary

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Chapters in this video

Transduction-Transplantation Mouse Model of Myeloproliferative Neoplasm

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Retroviral Transduction of T-cell Receptors in Mouse T-cells

Recombinant Retroviral Production and Infection of B Cells

Production of Replication-Defective Retrovirus by Transient Transfection of 293T cells

Generating De Novo Antigen-specific Human T Cell Receptors by Retroviral Transduction of Centric Hemichain

Streamlined Single Cell TCR Isolation and Generation of Retroviral Vectors for In Vitro and In Vivo Expression of Human TCRs

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Retroviral Overexpression of CXCR4 on Murine B-1a Cells and Adoptive Transfer for Targeted B-1a Cell Migration to the Bone Marrow and IgM Production

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation