Name: identification of homologous recombination events in mouse embryonic stem cells using southern blotting and polymerase chain reaction
Uploaded: 2018-11-20T13:30:00
Description: 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

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...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" style="width:903px;" width="903">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">BAC CLONE</td>
			<td style="width:232px;">BACPAC Resources Center (BPRC)</td>
			<td style="width:113px;">bMQ-330E21</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAGEN Large-Construct Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right">12462</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick Gel Extraction Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">28704</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAquick PCR Purification Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAprep Spin Miniprep Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">27104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAGEN Plasmid Plus Maxi Kit</td>
			<td style="width:232px;">QIAGEN</td>
			<td align="right" style="width:113px;">12963</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">PfuUltra High-Fidelity DNA Polymerase</td>
			<td style="width:232px;">Agilent</td>
			<td align="right" style="width:113px;">600382</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:391px;">T-easy vector</td>
			<td style="width:232px;">Promega</td>
			<td style="width:113px;">A1360</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:391px;">Nuclei Lysis Solution</td>
			<td style="width:232px;">Promega</td>
			<td style="width:113px;">A7941</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Precipitation Solution</td>
			<td style="width:232px;">Promega</td>
			<td>A7951</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Denaturing Solution</td>
			<td style="width:232px;">VWR</td>
			<td>351-013-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNA Neutralizing Solution</td>
			<td style="width:232px;">VWR</td>
			<td>351-014-131</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ready-To-Go DNA Labeling Beads (-dCTP)</td>
			<td style="width:232px;">VWR</td>
			<td>27-9240-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">UltraPure SSC, 20X</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">15557036</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">UltraPur Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v)</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">15593031</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">G418</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">10131035</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Salmon Sperm DNA Solution</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">15632011</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Platinu Taq DNA Polymerase High Fidelity</td>
			<td style="width:232px;">Thermo Fisher</td>
			<td align="right">11304029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:391px;">Not I</td>
			<td>Thermo Scientific</td>
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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 ...

This method can help answer key questions in molecular and cellular biology field, such as identifying homologous recombination events in mouse embryonic stem cells. The main advantage of this technique is that it is accurate, reliable, and widely used. So, this method can provide insight into identifying homologous recombination events in mouse embryonic stem cell.It can also be applied to other systems, such as genetically modified cell or animals. Generally, individual new to this method will struggle because it take long time and involves multiple steps. For each gDNA, mix 30 microliters of 10 x buffer for DRA one, three microliters of DRA one and 10 micrograms of gDNAs per sample.Add water until the final volume is 30 microliters, and incubate at 37 degrees celsius overnight to digest the gDNAs. Prepare a 1%agarose electrophoresis gel with ethidium bromide. Load the previously acquired samples along with a one KB ladder onto the gel, and run it at 30 to 40 volts overnight.The next day, soak the gel in a tray containing a 0.2 newton hydrochloric acid solution. Use a shaker to shake the tray gently for 20 minutes at room temperature. Transfer the gel to a tray containing a DNA denaturing solution.Shake it gently for 20 minutes at room temperature. Then transfer the gel into a tray containing a DNA neutralizing solution. And shake it gently for 20 minutes at room temperature.Using a rapid downward transfer system, transfer the DNAs from the gel to the membrane. Next, assemble the turble blotter and blotting stack as outlined in the instructions provided by the manufacturer. After this, take out the membrane and wash it with two X saline sodium citrate for one minute.Absorb the liquid with tissues, and then use a UV crosslinker to cross link the DNA with the membrane. To begin labeling the DNA probes with radioactivity, add the heat denatured probe DNAs to the tube containing the ready to go DNA labeling beads. Pipette up and down to mix.And add five microliters of radioactively labeled deoxycytodine triphosphate. Incubate at 37 degrees celsius for 15 minutes. Purify the labeled probes by using G 50 micro columns according to the instructions provided by the manufacturer.Use a scintillation counter to measure the radioactivity. To begin hybridizing the membranes with the labeled probes, place the membrane into the hybridization tube. Add the mixed pre hybridization solution.Place the tube into hybridization oven and let the pre hybridization proceed at 42 degrees celsius for 30 minutes. Then, remove the tube from the oven and pour the pre hybridization solution into a 50 milliliter tube. Add the denatured probe, and mix gently.To remove the non hybridized probes, place the membrane into a tray containing one X saline sodium citrate with 0.1%SDS and shake gently at 55 to 60 degrees celsius for 10 minutes. After this, wrap the membrane with plastic wrap and fix it in the exposure cassette. In a dark room, expose the membrane to two sheets of X ray film.Develop the film to visualize the results. Determine if a corresponding ES clone is the desired one with the target of recombination or not, according to the sizes of the DNA bands detected by the probes. In this study, southern blotting and PCR is utilized to identify HR events that occur in mouse ES cells for the generation of NM two genetic replacement mouse models using ES cells based HR mediated targeting technology.Because the genomic DNAs are cut into many fragments with different lengths, they display a smear like status on the DNA gel. This suggests a complete digestion of the genomic DNAs. As a final step of southern blotting, the signals of a radioactivity labeled probe hybridizing with a target DNA fragment are shown on the film.The appearance of expected bands reflects the occurrence of HR events in the ES clones. According to the predesign in the study, ES clones with mutated allele have two distinct size bands. While wild type ES clones only have one band, suggesting the desired ES clones are heterozygous.Following the PCR reaction, the PCR products can be analyzed on the DNA gel. If a specific NPCR band of the correct size is observed, this suggests the occurrence of HR events which can be further confirmed by sequencing that PCR band. The implication of this technique extended to the therapy of identifying point mutation because the foundation is identical.While attempting this procedure is important to remember to be patient and careful. Following this procedure, other method like microinjection of ES cell into blastocysts can be performed in order to answer additional question like there formation of chimeric animals. Aft its development, this technique paved the way for the researcher in their field of molecular and cellular biology to explore the function of a specific gene or the consequence of a mutated gene in mice or beyond.Don't forget that working with radioactivity labeler P32 DCTB can be extremely hazardous and precautions such as shielding with protection board should be always be taking while performing this procedure.

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