This work received support from the General Program of National Natural Science Foundation of China (Grants No. 31571432), the Human Provincial Natural Science Foundation of China (Grant No. 2015JC3097), and the Research Foundation of Education Bureau of Hunan Province, China (Grant No. 15K054).

1. Design of Targeting Construct(s), Probes for Southern Blot, and Primers for PCR
<ol>
	<li>Select the first coding exon (exon 2) of the Myh9 gene for disruption or insertion in the application of knockout/knock-in reported here.</li>
	<li>Retrieve the 5-kb upstream and 5-kb downstream DNA sequences surrounding the Myh9 exon 2 from the genome.ucsc.edu website.</li>
	<li>Analyze restriction digestion patterns of enzymes (REs) with 1&#8211;2 cuts in this 10-kb region by using pDRAW software to determine suitable RE(s) to digest the genomic DNA for Southern blotting. 
	NOTE: Dra I meets this requirement and is selected for the purpose.</li>
	<li>Select a 4 kb fragment immediate upstream of the Myh9 exon 2 as the left homology arm (LHA) and a 1.7 kb fragment immediate downstream sequence as the right homology arm (RHA); choose a 1 kb fragment 5&#39; upstream of the LHA as the left probe (LP) for Southern blotting and a 1.2 kb fragment 3&#39; downstream of the RHA as the right probe (RP), based on the above analysis.</li>
	<li>Use a primer3 program to design the forward and reverse primers for amplifying those four DNA fragments by PCR. Design a pair of primers with the forward primer resided near the 3&#39; terminal of a selection marker neomyocin resistance gene (P1) and the reverse primer located just outside the RHA (P2). 
	NOTE: This primer pair will be used for identifying targeted ES clones by PCR29.</li>
	<li>Find a 129Sv BAC clone covering mouse Myh9 gene locus by visiting the bacpac.chori.org website (note: isogenic DNA is preferred). Isolate BAC DNA using a kit suitable for purifying large pieces of DNA following the instructions provided by the manufacturer. 
	NOTE: Purified BAC DNA will be used as the template for PCR amplification.</li>
	<li>Draw a schematic representation of the targeting constructs, probes, and primers to summarize this information.</li>
</ol>
2. Generation of Targeting Construct(s) and Probes for Southern Blot, and the Preparation of Primers for PCR
<ol>
	<li>Order the PCR primers described above and dissolve them into a concentration of 20 &#181;M.</li>
	<li>Amplify the homology arms and probes by PCR in a reaction solution including 1 &#181;L forward and 1 &#181;L reverse primers, 1 &#181;L of BAC DNA (50 ng) as the template, 5 &#181;L of buffer for Pfu and 1 &#181;L of Pfu ultra as the DNA polymerase, and H2O up to 50 &#181;L. Perform the PCR in a PCR machine under the following conditions: 95 &#176;C for 3 min; 95 &#176;C for 30 s; 60 &#176;C for 30 s; 72 &#176;C and 1 kb/min; 30 cycles; finally, 72 &#176;C for 10 min.</li>
	<li>Purify PCR products using a PCR cleanup kit according to the protocol provided by the manufacturer and elute the DNA fragments with 40 &#181;L of H2O. Digest the purified PCR products with predesigned REs in a reaction solution containing 5 &#181;L of 10x buffer for REs, 2.5 &#181;L of RE1 and 2.5 &#181;L of RE2, and the eluted DNA, at 37 &#176;C for 2 h. Run a 1% DNA gel to separate target DNA bands, excise the gel containing the target DNA under the UV light, purify the target DNA fragments from the gel using a DNA gel extraction kit according to the protocol provided by the manufacturer, and elute the DNA fragments with 40 &#181;L of H2O.</li>
	<li>Clone the homology arms and replacement expression cassette(s) into the mpNTKV-LoxP vector according to the order of the amplified and purified RHA, LHA, and replacement expression cassette(s) released from other vectors to obtain the final targeting construct(s). Clone DNA fragments of the amplified and purified probes into the T-easy vector.</li>
	<li>Confirm the nucleotide sequences of all cloned DNA fragments by sequencing27,28.</li>
</ol>
3. Preparation of Targeting Construct(s), the Electroporation of ES Cells, and the Amplification of ES Clones
<ol>
	<li>Prepare each targeting construct using a plasmid maxiprep kit according to the protocol provided by the manufacturer. Linearize each construct plasmid by digesting it with Not I in a 400 &#181;L reaction including 40 &#181;L of 10x buffer for Not I, 10 &#181;L of Not I, 100 &#181;g of DNA, and H2O up to 400 &#181;L, at 37 &#176;C overnight.</li>
	<li>Purify the linearized targeting construct(s).
	<ol>
		<li>Extract the digested reaction solution 1x with an equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1) and centrifuge at a force of 2,000 x g for 10 min.</li>
		<li>Transfer the supernatant to a new 1.5-mL tube, precipitate the DNA using 2.5x ethanol and 0.1x 3M sodium acetate (pH 5.2) (volume ratio), and centrifuge at a force of 2,000 x g for 10 min.</li>
		<li>Remove the supernatant, wash the DNA pellet 1x with 1 mL of 75% ethanol, and centrifuge at a force of 2,000 x g for 5 min.</li>
		<li>Remove the supernatant and air-dry the DNA pellet for 5 min.</li>
		<li>Dissolve the linearized DNA pellet in sterile Tris-EDTA (TE) buffer at a final concentration of 1 &#181;g/&#181;L.</li>
	</ol>
	</li>
	<li>Mix 50 &#181;g of each linearized targeting construct with 0.5 x 107 ES cells. Perform the electroporation at 320 V and 250 &#181;F. Plate the electroporated ES cells onto dishes with neo-resistant MEF feeders.</li>
	<li>After 24 h, switch to ES cell medium with 400 &#181;g/mL G418 and 200 &#181;M ganciclovir and continue to culture for 4&#8211;5 days with a daily medium change. Pick up drug-resistant ES clones into 48-well plates. 
	NOTE: Normally, four 48-well plates are used per construct.</li>
	<li>Duplicate the 48-well plates. 
	NOTE: One set of the plates is cryopreserved, and the other set is used for genomic DNA preparation.</li>
</ol>
4. Preparation of Genomic DNAs and the Digestion with Restriction Enzyme(s)
<ol>
	<li>Prepare gDNAs from ES cells using a commercial kit (genomic DNA purification kit) with minor modifications.
	<ol>
		<li>Remove media from the ES cell culture and add 500 &#181;L of nuclei lysis solution, including RNaseA, directly to the wells to lyse the cells. 
		NOTE: The cell lysate can be stored at -80 &#176;C or treated immediately.</li>
		<li>Pipet up and down several times to lyse the cells fully and transfer them to a clean 1.5-mL tube.</li>
		<li>Add one-third of the volume of the protein precipitation solution to the 1.5 mL tube, vortex vigorously for 20 s, chill the samples on ice for 5 min, and then, centrifuge at a force of 2,000 x g for 5 min. Transfer the supernatant to another clean 1.5 mL tube containing an equal volume of isopropanol; gently mix the solution. (Note that white thread-like strands can be seen at this moment.) Centrifuge at a force of 2,000 x g for 1 min; then, discard the supernatant.</li>
		<li>Wash the gDNA pellet with 1 mL of 70% ethanol at room temperature, centrifuge at a force of 2,000 x g for 1 min, aspirate the supernatant carefully, and then, air-dry the gDNA pellet for 3 min.</li>
		<li>Dissolve the gDNAs with 100 &#181;L of DNA rehydration solution and, then, incubate at 65 &#176;C for 1 h or at 4 &#176;C overnight.</li>
		<li>Store the gDNAs at 2&#8211;8 &#176;C.</li>
	</ol>
	</li>
	<li>Digest the gDNAs with predesigned RE Dra I. Set up a 30-&#181;L digestion reaction by mixing 3 &#181;L of 10x buffer for Dra I, 3 &#181;L of Dra I, 10 &#181;g of gDNAs/sample, and H2O up to 30 &#181;L, and incubate at 37 &#176;C overnight.</li>
	<li>Check the completeness of the digestion by DNA gel, analyzing 5 &#181;L of the digested reaction, and then, add the 3 &#181;L of 10x DNA-loading buffer for the subsequent step.</li>
</ol>
5. Southern Blotting and PCR Identification
<ol>
	<li>Southern blotting screening
	<ol>
		<li>Separate the digested gDNAs by electrophoresis and transfer to a membrane.
		<ol>
			<li>Prepare a 1% agarose electrophoresis gel with ethidium bromide (EB), load the samples from step 4.3 and a 1 kb ladder, and run the gel with a low voltage (30&#8211;40 V) overnight.</li>
			<li>Take out the gel and take a picture with a DNA gel-imaging system after electrophoresis. Check whether the digested and separated gDNAs display a smear-like image.</li>
			<li>Soak the gel in a tray with 0.2 N HCl solution and shake it gently for 20 min at room temperature.</li>
			<li>Transfer the gel to DNA-denaturing solution and shake it gently for 20 min at room temperature.</li>
			<li>Switch the gel into DNA-neutralizing solution and shake it gently for 20 min at room temperature. 
			NOTE: The gel is prone to breakage after this step, so it must be handled carefully.</li>
			<li>Use the rapid downward transfer system to transfer the DNAs from the gel to the membrane. Assemble the TurboBlotter and blotting stack according to the instructions provided by the manufacturer. 
			NOTE: 10x or 20x saline-sodium citrate (SSC) solution is used as a transfer buffer. In general, 3 h of transfer is enough to transfer 95% of gDNAs from gel to membrane; however, a longer time of transfer is innocuous.</li>
			<li>Take out the membrane and wash it with 2x SSC for 1 min, absorb the liquid with tissues, and then cross-link the DNA with the membrane using a UV crosslinker. 
			NOTE: The membrane can be stored at 4 &#176;C for one week.</li>
		</ol>
		</li>
		<li>Label the DNA probes with radioactivity.
		<ol>
			<li>Purify the probe plasmids using a miniprep kit according to the protocol provided by the manufacturer.</li>
			<li>Release the DNA fragments of the probes from the plasmid vector by EcoR I digestion in a reaction solution including 5 &#181;L of buffer for EcoR I, 2 &#181;L of EcoR I enzyme, 20 &#181;g of plasmid DNA, and H2O up to 50 &#181;L, for 2 h.</li>
			<li>Run a 1% DNA gel for separating the probe DNA fragments from the vector and purify the DNA fragments of the probes with a DNA gel extraction kit according to the protocol provided by the manufacturer.</li>
			<li>Using 1 &#181;L of DNA solution, measure the DNA concentration of the probe DNA fragments with a spectrophotometer at a wavelength of 260/280 nm.</li>
			<li>Prepare 40-ng of probe DNAs in a 1.5 mL tube with 45 &#181;L of TE buffer, boil for 3 min, spin briefly, and then, place the tube(s) on ice for 2 min.</li>
			<li>Add the heat-denatured probe DNAs to the tube containing ready-to-go DNA-labeling beads (-dCTP), pipet up and down to mix, add 5 &#181;L of [&#945;32P]dCTP, and then, incubate at 37 &#176;C for 15 min.</li>
			<li>Purify the labeled probes by using G-50 microcolumns according to the instructions provided by the manufacturer and, then, measure the radioactivity by a scintillation counter (optional).</li>
		</ol>
		</li>
		<li>Hybridize the membrane(s) with the labeled probes.
		<ol>
			<li>Prehybridize the membrane.
			<ol>
				<li>Prewarm the hybridization solution at 42 &#176;C for 30 min. Mix 20 mL of prewarmed hybridization solution with 200 &#181;g of boiled salmon sperm DNA in a 50-mL tube.</li>
				<li>Place the membrane into the hybridization tube. Add the mixed prehybridization solution to the hybridization tube. Place it into the hybridization oven (set rolling and the temperature at 42 &#176;C) and let the prehybridization proceed for 30 min.</li>
			</ol>
			</li>
			<li>Hybridize the membrane with the labeled probe(s).
			<ol>
				<li>Take out the hybridization tube and pour the prehybridization solution into a 50 mL tube; add the denatured probe (heated at 100 &#176;C for 3 min) from step 5.1.2.7 to this tube and mix gently. 
				NOTE: Reduce any inducing bubbles.</li>
				<li>Return the mixed solution to the hybridization tube and perform the hybridization at 42 &#176;C overnight.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Wash the membrane(s) to remove nonhybridized probes.
		<ol>
			<li>Place the membrane(s) into a tray with 1x SSC + 0.1% SDS and shake gently at 55&#8211;60 &#176;C for 10 min.</li>
			<li>Transfer the membrane(s) to a tray with 0.5x SSC + 0.1% SDS and shake gently at 55&#8211;60 &#176;C for 10 min.</li>
			<li>Check the radioactivity on the membrane(s) by using a portable Geiger counter to decide whether a third washing is required.</li>
		</ol>
		</li>
		<li>Expose the radioactivity on the membrane to X-ray films.
		<ol>
			<li>Remove the liquid from the washed membrane(s).</li>
			<li>Enfold the membrane(s) with plastic wrap and fix it/them in the exposure cassette.</li>
			<li>Expose the membrane to two sheets of X-ray film in a dark room.</li>
			<li>Place the exposure cassette at -80 &#176;C overnight or longer.</li>
		</ol>
		</li>
		<li>Develop the films to visualize the results. Evaluate whether a corresponding ES clone is the desired one with the targeted recombination or not, according to the sizes of the DNA bands detected by the probes.</li>
		<li>Rehybridize the same membrane by another probe after stripping off the used probe according to the following procedure: take out the used membrane, wash it 1x with clean H2O, and then, incubate it in striping solution (55% formamide, 2% SSPE, 1% SDS, H2O) at 65 &#176;C with gentle shaking for 1&#8211;2 h.</li>
	</ol>
	</li>
	<li>PCR identification
	<ol>
		<li>Perform PCR identification of the desired ES clones in a 50-&#181;L reaction solution including 5 &#181;L of 10x PCR buffer, 2 &#181;L of 50 mM MgSO4, 1 &#181;L of 10 mM dNTP, 1 &#181;L of 20 &#181;M forward primer, 1 &#181;L of 20 &#181;M reverse primer, 1 &#181;L of high-fidelity platinum Taq, gDNAs (~100 ng), and H2O up to 50 &#181;L.</li>
		<li>Use the following PCR reaction conditions: an initial denaturation at 94&#176;C for 3 min, 30 cycles of denaturation at 94&#176;C for 30 s, annealing at 60&#176;C for 30 s and an extension at 68&#176;C for 132 s, and a final step of 68&#176;C for 10 min.</li>
		<li>Analyze the PCR products by electrophoresis in 1.0% agarose.</li>
		<li>Clone the PCR fragments with their expected size into the T-easy vector and sequence to confirm the presence of a partial sequence of the target vector.</li>
	</ol>
	</li>
</ol>

<ol><li>Gao, G., McMahon, C., Chen, J., Rong, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+powerful+method+combining+homologous+recombination+and+site-specific+recombination+for+targeted+mutagenesis+in+Drosophila%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">A powerful method combining homologous recombination and site-specific recombination for targeted mutagenesis in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (37), 13999-14004 (2008).</li>
<li>Skarnes, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+conditional+knockout+resource+for+the+genome-wide+study+of+mouse+gene+function%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A conditional knockout resource for the genome-wide study of mouse gene function.</a> Nature. 474 (7351), 337-342 (2011).</li>
<li>Vogel, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nobel+Prizes.+A+knockout+award+in+medicine%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">Nobel Prizes. A knockout award in medicine.</a> Science. 318 (5848), 178-179 (2007).</li>
<li>Salsman, J., Dellaire, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Precision+genome+editing+in+the+CRISPR+era%5BTitle%5D)+AND+%22Biochemistry.+Cell+Biology%22%5BJournal%5D)">Precision genome editing in the CRISPR era.</a> Biochemistry. Cell Biology. 95 (2), 187-201 (2017).</li>
<li>Capecchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+targeting+in+mice%3A+functional+analysis+of+the+mammalian+genome+for+the+twenty-first+century%5BTitle%5D)+AND+%22Nature+Reviews+Genetics%22%5BJournal%5D)">Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century.</a> Nature Reviews Genetics. 6 (6), 507-512 (2005).</li>
<li>Van, dW. L., Adams, D. J., Bradley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tools+for+targeted+manipulation+of+the+mouse+genome%5BTitle%5D)+AND+%22Physiological+Genomics%22%5BJournal%5D)">Tools for targeted manipulation of the mouse genome.</a> Physiological Genomics. 11 (3), 133-164 (2002).</li>
<li>Glaser, S., Anastassiadis, K., Stewart, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+issues+in+mouse+genome+engineering%5BTitle%5D)+AND+%22Nature+Genetics%22%5BJournal%5D)">Current issues in mouse genome engineering.</a> Nature Genetics. 37 (11), 1187(2005).</li>
<li>Bradley, A., Evans, M., Kaufman, M. H., Robertson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Formation+of+germ-line+chimaeras+from+embryo-derived+teratocarcinoma+cell+lines%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.</a> Nature. 309 (5965), 255-256 (1984).</li>
<li>Robertson, E., Bradley, A., Kuehn, M., Evans, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Germ-line+transmission+of+genes+introduced+into+cultured+pluripotential+cells+by+retroviral+vector%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector.</a> Nature. 323 (6087), 445-448 (1986).</li>
<li>Thomas, K. R., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Site-directed+mutagenesis+by+gene+targeting+in+mouse+embryo-derived+stem+cells%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells.</a> Cell. 51 (3), 503-512 (1987).</li>
<li>Skarnes, W. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+conditional+knockout+resource+for+the+genome-wide+study+of+mouse+gene+function%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A conditional knockout resource for the genome-wide study of mouse gene function.</a> Nature. 474 (7351), 337(2011).</li>
<li>Collins, F. S., Rossant, J., Wurst, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+mouse+for+all+reasons%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">A mouse for all reasons.</a> Cell. 128 (1), 9-13 (2007).</li>
<li>Poueymirou, W. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((F0+generation+mice+fully+derived+from+gene-targeted+embryonic+stem+cells+allowing+immediate+phenotypic+analyses%5BTitle%5D)+AND+%22Nature+Biotechnology%22%5BJournal%5D)">F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses.</a> Nature Biotechnology. 25 (1), 91-99 (2007).</li>
<li>Pettitt, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Agouti+C57BL%2F6N+embryonic+stem+cells+for+mouse+genetic+resources%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Agouti C57BL/6N embryonic stem cells for mouse genetic resources.</a> Nature Methods. 6 (7), 493-495 (2009).</li>
<li>Gertsenstein, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Efficient+Generation+of+Germ+Line+Transmitting+Chimeras+from+C57BL%2F6N+ES+Cells+by+Aggregation+with+Outbred+Host+Embryos%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Efficient Generation of Germ Line Transmitting Chimeras from C57BL/6N ES Cells by Aggregation with Outbred Host Embryos.</a> PLoS One. 5 (6), 11260(2012).</li>
<li>Smithies, O., Gregg, R. G., Boggs, S. S., Koralewski, M. A., Kucherlapati, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Insertion+of+DNA+sequences+into+the+human+chromosomal+%7C%5Bbeta%5D%7C-globin+locus+by+homologous+recombination%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Insertion of DNA sequences into the human chromosomal |[beta]|-globin locus by homologous recombination.</a> Nature. 317 (6034), 230-234 (1985).</li>
<li>Deng, C., Capecchi, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reexamination+of+gene+targeting+frequency+as+a+function+of+the+extent+of+homology+between+the+targeting+vector+and+the+target+locus%5BTitle%5D)+AND+%22Molecular+%26+Cellular+Biology%22%5BJournal%5D)">Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus.</a> Molecular & Cellular Biology. 12 (8), 3365(1992).</li>
<li>Lay, J. M., Friishansen, L., Gillespie, P. J., Samuelson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+confirmation+of+gene+targeting+in+embryonic+stem+cells+using+two+long-range+PCR+techniques%5BTitle%5D)+AND+%22Transgenic+Research%22%5BJournal%5D)">Rapid confirmation of gene targeting in embryonic stem cells using two long-range PCR techniques.</a> Transgenic Research. 7 (2), 135-140 (1998).</li>
<li>Langerak, P., Nygren, A. O. H., Schouten, J. P., Jacobs, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+quantitative+detection+of+homologous+and+non-homologous+recombination+events+using+three+oligonucleotide+MLPA%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA.</a> Nucleic Acids Research. 33 (22), 188(2005).</li>
<li>Gómezrodríguez, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advantages+of+q-PCR+as+a+method+of+screening+for+gene+targeting+in+mammalian+cells+using+conventional+and+whole+BAC-based+constructs%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs.</a> Nucleic Acids Research. 36 (18), 117(2008).</li>
<li>Kim, H. S., Smithies, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recombinant+fragment+assay+for+gene+targeting+based+on+the+polymerase+chain+reaction%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Recombinant fragment assay for gene targeting based on the polymerase chain reaction.</a> Nucleic Acids Research. 16 (18), 8887-8903 (1988).</li>
<li>Joyner, A. L., Skarnes, W. C., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+a+mutation+in+mouse+En-2+gene+by+homologous+recombination+in+embryonic+stem+cells%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Production of a mutation in mouse En-2 gene by homologous recombination in embryonic stem cells.</a> Nature. 338 (6211), 153-156 (1989).</li>
<li>Ma, X., Adelstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+vertebrate+nonmuscle+Myosin+II+in+development+and+human+disease%5BTitle%5D)+AND+%22Bioarchitecture%22%5BJournal%5D)">The role of vertebrate nonmuscle Myosin II in development and human disease.</a> Bioarchitecture. 4 (3), 88-102 (2014).</li>
<li>Malonek, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Relationships+between+the+dynamics+of+cortical+blood+flow%2C+oxygenation%2C+and+volume+changes+following+sensory+stimulation%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Relationships between the dynamics of cortical blood flow, oxygenation, and volume changes following sensory stimulation.</a> Proceedings of the National Academy of Sciences of the United States of America. 94, (1997).</li>
<li>Takeda, K., Kishi, H., Ma, X., Yu, Z. X., Adelstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ablation+and+mutation+of+nonmuscle+myosin+heavy+chain+II-B+results+in+a+defect+in+cardiac+myocyte+cytokinesis%5BTitle%5D)+AND+%22Circulation+Research%22%5BJournal%5D)">Ablation and mutation of nonmuscle myosin heavy chain II-B results in a defect in cardiac myocyte cytokinesis.</a> Circulation Research. 93 (4), 330-337 (2003).</li>
<li>Conti, M. A., Evenram, S., Liu, C., Yamada, K. M., Adelstein, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Defects+in+cell+adhesion+and+the+visceral+endoderm+following+ablation+of+nonmuscle+myosin+heavy+chain+II-A+in+mice%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice.</a> Journal of Biological Chemistry. 279 (40), 41263-41266 (2004).</li>
<li>Wang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nonmuscle+myosin+II+isoform+and+domain+specificity+during+early+mouse+development%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Nonmuscle myosin II isoform and domain specificity during early mouse development.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (33), 14645-14650 (2010).</li>
<li>Zhang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+MYH9-related+disease%3A+mutations+in+nonmuscle+myosin+II-A%5BTitle%5D)+AND+%22Blood%22%5BJournal%5D)">Mouse models of MYH9-related disease: mutations in nonmuscle myosin II-A.</a> Blood. 119 (1), 238(2012).</li>
<li>Liu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Identification+and+characterization+of+MYH9+locus+for+high+efficient+gene+knock-in+and+stable+expression+in+mouse+embryonic+stem+cells%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">Identification and characterization of MYH9 locus for high efficient gene knock-in and stable expression in mouse embryonic stem cells.</a> PLoS One. 13 (2), 0192641(2018).</li>
<li>Saito, S., Adachi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advances+in+the+Development+of+Gene-Targeting+Vectors+to+Increase+the+Efficiency+of+Genetic+Modification%5BTitle%5D)+AND+%22Biological+%26+Pharmaceutical+Bulletin%22%5BJournal%5D)">Advances in the Development of Gene-Targeting Vectors to Increase the Efficiency of Genetic Modification.</a> Biological & Pharmaceutical Bulletin. 39 (1), 25-32 (2016).</li>
<li>Langerak, P., Nygren, A., Schouten, J., Jacobs, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+and+quantitative+detection+of+homologous+and+non-homologous+recombination+events+using+three+oligonucleotide+MLPA%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Rapid and quantitative detection of homologous and non-homologous recombination events using three oligonucleotide MLPA.</a> Nucleic Acids Research. 33 (22), 188(2005).</li>
<li>Martin, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single+amino+acid+substitution+in+ORF1+dramatically+decreases+L1+retrotransposition+and+provides+insight+into+nucleic+acid+chaperone+activity%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">A single amino acid substitution in ORF1 dramatically decreases L1 retrotransposition and provides insight into nucleic acid chaperone activity.</a> Nucleic Acids Research. 36 (18), 5845-5854 (2008).</li>
<li>Kamisugi, Y., Cuming, A. C., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Parameters+determining+the+efficiency+of+gene+targeting+in+the+moss+Physcomitrella+patens%5BTitle%5D)+AND+%22Nucleic+Acids+Research%22%5BJournal%5D)">Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.</a> Nucleic Acids Research. 33 (19), 173(2005).</li>
<li>Luo, Y., Bolund, L., Sørensen, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pig+gene+knockout+by+rAAV-mediated+homologous+recombination%3A+comparison+of+BRCA1+gene+knockout+efficiency+in+Yucatan+and+G%C3%B6ttingen+fibroblasts+with+slightly+different+target+sequences%5BTitle%5D)+AND+%22Transgenic+Research%22%5BJournal%5D)">Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Göttingen fibroblasts with slightly different target sequences.</a> Transgenic Research. 21 (3), 671-676 (2012).</li>
<li>Croning, M. D., Fricker, D. G., Komiyama, N. H., Grant, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Automated+design+of+genomic+Southern+blot+probes%5BTitle%5D)+AND+%22BMC+Genomics%22%5BJournal%5D)">Automated design of genomic Southern blot probes.</a> BMC Genomics. 11 (1), 74(2010).</li>
<li>Zimmer, A., Gruss, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Production+of+chimaeric+mice+containing+embryonic+stem+%28ES%29+cells+carrying+a+homoeobox+Hox+1.1+allele+mutated+by+homologous+recombination%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Production of chimaeric mice containing embryonic stem (ES) cells carrying a homoeobox Hox 1.1 allele mutated by homologous recombination.</a> Nature. 338 (6211), 150-153 (1989).</li>
</ol>

The authors have nothing to disclose.

Currently, designer nucleases for genome editing still cannot replace ES cell-based gene-targeting technology due to its issues of off-target effects, and difficulty in inserting a long DNA fragment30,31. As the golden methods for identifying HR events that occurred in mouse ES cells, this report provides a detailed protocol of Southern blotting and PCR for the field. We validated the reliability of these methods by analyzing individual clones from mouse ES cells...

The technology of gene targeting by HR in murine ES cells provides a powerful tool for dissecting the cellular consequences of specific genetic mutations1,2. The importance and significance of this technology are reflected in its recognition by the 2007 Nobel Prize in Physiology or Medicine3,4; meanwhile, it represents the advent of the modern era of gene engineering5. Gene targeting through HR can be utilized to engineer virtually any alteration ranging from point mutations to large chromosomal rearrangements in the genome of mouse ES cells6,7. It is well known that, before the emergence of so-called genome editing tools, the generation of a gene knockout mouse required the application of gene-targeting technology in ES cells8,9,10. During the past two decades, more than 5,000 gene-targeted mice were produced by this approach for modeling human diseases or studying gene functions11. A genome-wide knockout effort has been established for distributing gene-targeting vectors, targeted ES cell clones, and live mice to the scientific community2,12,13,14,15. Undoubtedly, ES cell-based HR-mediated gene-targeting technology has greatly advanced our understanding of the functions of genes played in physiological or pathological context.
Because HR is a relatively infrequent event in mammalian cells16,17, the important and next step following gene targeting in murine ES cells is to analyze numerous ES colonies for identifying a few clones with mutations resulting from HR with the targeting vector18. The gold methods for identifying HR events include Southern blotting and PCR19,20. The advantages of the approaches include that Southern blotting can identify correctly targeted ES clones and allows researchers to analyze the structure of the gene-targeted event, such as a verification of a single copy insertion of the construct, while a PCR-based strategy permits more rapid screening for HR events21,22. Though these methods have drawbacks, such as that they are time-consuming and can have false positives, the combinational usage of them is widely accepted and applied by most labs in identifying HR events, particular in ES cells, for generating genetically modified animals.
Three isoforms of nonmuscle myosin II (NM II) in mammals, each consisting of two identical NMHC IIs which are encoded by three different genes (named Myh9, Myh10, and Myh14) and two pairs of light chains, are referred to as NM II-A, II-B, and II-C23. Previous studies have indicated that at least the isoforms of NM II-A and II-B are essential for mouse development because the in vivo ablation of these isoforms results in embryonic lethality24,25,26. To circumvent this problem and obtain novel insights into the isoform-specific functions of NM II-A and II-B in the later stages of mouse development, a genetic replacement strategy using ES cell-based HR-mediated gene-targeting technology was adopted to generate a series of mouse models27. In the course of identifying the desired ES clones, both Southern blotting and PCR methods were utilized, and these proved to be efficient and reliable27,28.
This paper intends to provide a detailed description of Southern blotting and PCR, including the design of targeting vector(s), probe(s), and primers, and the execution of experiments, as well as the analysis of results exemplified by identifying HR event occurrence in ES cells for creating genetic replacement NM II mouse models and representative data. The protocols of these two methods presented here can also be adopted for identifying the genotypes of genetically modified cells or animals.

<table><tbody><tr><td>BAC CLONE</td><td>BACPAC Resources Center (BPRC)</td><td>bMQ-330E21</td><td></td></tr><tr><td>QIAGEN Large-Construct Kit</td><td>QIAGEN</td><td>12462</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>QIAGEN</td><td>28104</td><td></td></tr><tr><td>QIAprep Spin Miniprep Kit</td><td>QIAGEN</td><td>27104</td><td></td></tr><tr><td>QIAGEN Plasmid Plus Maxi Kit</td><td>QIAGEN</td><td>12963</td><td></td></tr><tr><td>PfuUltra High-Fidelity DNA Polymerase</td><td>Agilent</td><td>600382</td><td></td></tr><tr><td>T-easy vector</td><td>Promega</td><td>A1360</td><td></td></tr><tr><td>Nuclei Lysis Solution</td><td>Promega</td><td>A7941</td><td></td></tr><tr><td>Protein Precipitation Solution</td><td>Promega</td><td>A7951</td><td></td></tr><tr><td>DNA Denaturing Solution</td><td>VWR</td><td>351-013-131</td><td></td></tr><tr><td>DNA Neutralizing Solution</td><td>VWR</td><td>351-014-131</td><td></td></tr><tr><td>Ready-To-Go DNA Labeling Beads (-dCTP)</td><td>VWR</td><td>27-9240-01</td><td></td></tr><tr><td>UltraPure SSC, 20x</td><td>Thermo Fisher</td><td>15557036</td><td></td></tr><tr><td>UltraPur Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td><td>Thermo Fisher</td><td>15593031</td><td></td></tr><tr><td>G418</td><td>Thermo Fisher</td><td>10131035</td><td></td></tr><tr><td>Salmon Sperm DNA Solution</td><td>Thermo Fisher</td><td>15632011</td><td></td></tr><tr><td>Platinu Taq DNA Polymerase High Fidelity</td><td>Thermo Fisher</td><td>11304029</td><td></td></tr><tr><td>Not I</td><td>Thermo Scientific</td><td>ER0592</td><td></td></tr><tr><td>Dra I</td><td>Thermo Scientific</td><td>ER0221</td><td></td></tr><tr><td>EcoR I</td><td>Thermo Scientific</td><td>ER0271</td><td></td></tr><tr><td>Ganciclovir</td><td>Sigma</td><td>G2536</td><td></td></tr><tr><td>Whatman&amp;nbsp;TurboBlotter Transfer System, Large Kits</td><td>Fisher Scientific</td><td>09-301-188</td><td></td></tr><tr><td>[&amp;alpha;&lt;sup&gt;32&lt;/sup&gt;P]dCTP</td><td>PerkinElmer</td><td>NEG013H100UC</td><td></td></tr><tr><td>ProbeQuan G-50 Micro Columns</td><td>GE Healthcare</td><td>28-9034-08</td><td></td></tr><tr><td>Hybrisol I Hybridization Solution</td><td>Millipore</td><td>S4040</td><td></td></tr><tr><td>Kodak X-Ray Film</td><td>Z&amp;amp;Z Medical</td><td>844 5702</td><td></td></tr></tbody></table>

identification of homologous recombination events in mouse embryonic stem cells using southern blotting and polymerase chain reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

In this paper, a detailed protocol of Southern blotting and PCR is described, which is utilized to identify HR events that occurred in mouse ES cells for the generation of NM II genetic replacement mouse models, using ES cells-based HR-mediated targeting technology. Though Southern blotting and PCR, as well as traditional gene-targeting technology, have been widely used for several decades, the successful application of them needs to be planned carefully. At least these aspects are requir...

Watch this Scientific Journal Video about Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction at JoVE.com

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

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9.3K Views.  Hunan Agricultural University (HUNAU). Deze methode kan helpen bij het beantwoorden van belangrijke vragen in moleculaire en cellulaire biologie veld, zoals het identificeren van homologe recombinatie gebeurtenissen in de muis embryonale stamcellen. Het belangrijkste voordeel van deze techniek is dat het nauwkeurig, betrouwbaar en veel gebruikt is. Deze methode kan dus inzicht geven in het identificeren van homologe recombinatiegebeurtenissen in de embryonale stamcel van muizen.Het kan ook worden toegepast op andere systeme...

Video: Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Developmental Biology

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

Identifying DNA Damage in Prophase-Arrested Oocytes of Mice

Detection of DNA Double-Stranded Breaks in Mouse Oocytes

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain arrested at the prophase of meiosis I. The quiescent state may last for years until the oocytes receive a stimulus to grow and obtain the competency to resume meiosis. This protracted state of arrest makes them extremely susceptible to accumulating DNA-damaging insults, which affect the genetic integrity of the female gametes and, therefore, the genetic integrity of the future embryo. 
Consequently, the development of an accurate method to detect DNA damage, which is the first step for the establishment of DNA damage response mechanisms, is of vital importance. This paper describes a common protocol to test the presence and progress of DNA damage in prophase-arrested oocytes during a period of 20 h. Specifically, we dissect mouse ovaries, retrieve the cumulus-oocyte complexes (COCs), remove the cumulus cells from the COCs, and culture the oocytes in &#924;2 medium containing 3-isobutyl-1-methylxanthine to maintain the state of arrest. Thereafter, the oocytes are treated with the cytotoxic, antineoplasmic drug, etoposide, to engender double-strand breaks (DSBs). 
By using immunofluorescence and confocal microscopy, we detect and quantify the levels of the core protein &#947;H2AX, which is the phosphorylated form of the histone H2AX. H2AX becomes phosphorylated at the sites of DSBs after DNA damage. The inability to restore DNA integrity following DNA damage in oocytes can lead to infertility, birth defects, and increased rates of spontaneous abortions. Therefore, the understanding of DNA damage response mechanisms and, at the same time, the establishment of an intact method for studying these mechanisms are essential for reproductive biology research.

Author Spotlight: Understanding DNA Damage Response in Mammalian Oocytes and Preimplantation Embryos 

Maintaining oocyte genome integrity is necessary to ensure the genetic fidelity in the resulting embryo. Here, we present an accurate protocol for detecting DNA double-strand breaks in mammalian female germ cells.

Oocytes are amongst the biggest and most long-lived cells in the female body. They are formed in the ovaries during embryonic development and remain ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

Biology

Comparative Analysis of Forward and Reverse Transfection Methods on mPSCs

An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Author Spotlight: Advancements in Synthetic Genetic Devices for Stem Cell Fate Manipulation and Cellular Therapy Development

The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical ...

Isolation and Genotyping of Gastrulating Mouse Embryo for Single-Cell Sequencing

Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Author Spotlight: A Pipeline to Analyze Lineage-Specific Mutant Embryos at Single-Cell Resolution

This paper establishes a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states ...

Recombineering Homologous Recombination Constructs in Drosophila

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a genetic model organism for human-disease related research. Recent years have seen technical advancements like homologous recombination and recombineering. However, generating unequivocal null mutations or tagging endogenous proteins remains a substantial effort for most genes. Here, we describe and demonstrate techniques for using recombineering-based cloning methods to generate vectors that can be used to target and manipulate endogenous loci in vivo. Specifically, we have established a combination of three technologies: (1) BAC transgenesis/recombineering, (2) ends-out homologous recombination and (3) Gateway technology to provide a robust, efficient and flexible method for manipulating endogenous genomic loci. In this protocol, we provide step-by-step details about how to (1) design individual vectors, (2) how to clone large fragments of genomic DNA into the homologous recombination vector using gap repair, and (3) how to replace or tag genes of interest within these vectors using a second round of recombineering. Finally, we will also provide a protocol for how to mobilize these cassettes in vivo to generate a knockout, or a tagged gene via knock-in. These methods can easily be adopted for multiple targets in parallel and provide a means for manipulating the Drosophila genome in a timely and efficient manner.

Recombineering Homologous Recombination Constructs in Drosophila

Homologous recombination techniques greatly advance Drosophila genetics by enabling the creation of molecularly precise mutations. The recent adoption of recombineering allows one to manipulate large pieces of DNA and transform them into Drosophila6. The methods presented here combine these techniques to rapidly generate large homologous recombination vectors.

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

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