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Abstract

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Protocol

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Acknowledgements

Materials

References

Genetics

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

Published: November 20th, 2018

DOI:

10.3791/58467

1Lab of Animal Models and Functional Genomics (LAMFG), The Key Laboratory of Animal Vaccine & Protein Engineering, College of Veterinary Medicine, Hunan Agricultural University (HUNAU), 2Department of Pathology, Georgetown University Medical School, 3College of Food Science and Technology, Hunan Agricultural University (HUNAU), 4Lab of Molecular Cardiology (LMC), National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH), 5Transgenic Core, National Heart, Lung, and Blood Institute (NHLBI)/National Institutes of Health (NIH)
* These authors contributed equally

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 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.

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 m....

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1. Design of Targeting Construct(s), Probes for Southern Blot, and Primers for PCR

  1. Select the first coding exon (exon 2) of the Myh9 gene for disruption or insertion in the application of knockout/knock-in reported here.
  2. Retrieve the 5-kb upstream and 5-kb downstream DNA sequences surrounding the Myh9 exon 2 from the genome.ucsc.edu website.
  3. Analyze restriction digestion patterns of enzymes (REs) with 1–2 cuts in this 10-kb region by using pDRAW software to determine.......

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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.......

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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.......

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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).

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

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