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

Summary

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.

Abstract

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.

Introduction

The technology of gene targeting by HR in murine ES cells provides a powerful tool for dissecting the cellular consequences of specific genetic mutations^1,2. The importance and significance of this technology are reflected in its recognition by the 2007 Nobel Prize in Physiology or Medicine^3,4; 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....

Protocol

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 suitable RE(s) to digest the genomic DNA for Southern blotting.
   NOTE: Dra I meets this requirement and is selected for the purpose.
4. ....

Results

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.......

Discussion

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 fragment^30,31. 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.......

Materials

List of materials used in this article

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
[α32P]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