ES Cell-Mediated Gene Targeting

Introduction

Note: If you want a transgene targeted to the ROSA26 or H11 loci, please see our Targeted Transgenesis service description.

Gene targeting is the process of disrupting or mutating a specific genetic locus in embryonic stem (ES) cells, usually with the intention of making knock-out or knock-in mice.  Other methods of mutating the mouse genome include chemical mutagenesis, insertion of viral vectors or transposons, and random integration of gene-trap or promoter-trap vectors. Use of Cas9 nuclease is probably the most popular method to mediate genome engineering directly in mouse embryos, without the need to use ES cells. Note, CRISPR engineering can also be used in ES cells to engineer changes in the genome, including insertion or substitution of relatively large regions of DNA.

The targeting construct is usually a plasmid that contains two long stretches of genomic DNA, called homology arms, which are designed to match as closely as possible the genomic DNA of the ES cell line being targeted. These arms drive the homologous recombination event that results in insertion of the construct into the desired locus.  Bacterial artificial chromosomes (BACs) can also be used as targeting vectors.

It is the client’s responsibility to design and engineer the targeting construct. See our tips on targeting construct design. We can recommend outside sources of construct engineering services, including recombineering. Please feel free to contact the TMF for advice on the design of targeting constructs for use in ES cells.

The entire gene targeting process consists of the following major steps. Each of these steps can be ordered as a separate service from our facility.

1) Linearize the targeting construct; introduce it into ES cells by electroporation; grow clones under positive selection; pick several hundred resistant clones into 96-well plates; split clones into duplicate sets; freeze one set and extract DNA from the other set.

2) Genotype all clones by Southern blot using a probe specific for one end of the inserted DNA. PCR is usually less suitable for this step. The probe sequence must lie outside of the homology arms, and probes must be tested by the client prior to electroporation of the targeting construct.

3) Expand clones that have the correct genotype (heterozygous for the targeted allele), freeze back multiple vials of each clone, and prepare about 50 micrograms of genomic DNA from each clone for a second Southern blot to characterize the other end of the inserted DNA.  We can perform the second Southern blot for an additional fee.

We recommend doing a third Southern blot with a probe specific for the selection cassette in the targeting construct, to rule out the possibility that multiple copies of the construct were inserted.  We can do this Southern blot for an additional fee. Clones that pass all 3 assays should have their average chromosome number determined and, if they are at least 70% euploid, they may then be used for injection into blastocysts to make chimeric mice (see our ES cell injection service).

Additional in vitro manipulations may be desired if a conditional targeting construct is being used (see below). We offer a secondary electroporation service in which we transfect an ES cell clone with a recombinase-expressing plasmid, pick two 96-well plates of clones, and extract DNA from them, which the client then analyzes for the desired recombination event.  However, we recommend that all Cre or Flp recombinations be done in vivo to avoid the additional time in culture that may compromise the ability of the ES cells to "go germline."

We normally produce chimeric mice by injecting an ES cell clone into mouse blastocysts. We also have the capability of making chimeras by aggregation, but this will not work for some ES cell lines.  A chimera is said to "go germline" when some of its offspring arise from ES cell-derived gametes.  The ability of ES cells to contribute to the germline of a chimeric mouse is highly dependent on culture conditions, the total number of passages (cycles of splitting or freezing), and the degree of aneuploidy.  Most targeted clones are heterozygous for the targeted allele and therefore ES cell-derived gametes have only a 50% chance of carrying the targeted allele.  Clones with <50% euploid cells can produce chimeras but will most likely NOT go germline.