Pooling and PCR as a method to combat low frequency gene targeting in mouse embryonic stem cells

Abstract

The introduction of germ line modifications by gene targeting in mouse embryonic stem (ES) cells has proven a fundamental technology to relate genes to mammalian biology. Critical aspects required for successful gene targeting have traditionally been experimental enhancements that increase the frequency or detection of homologous recombination within ES cells; however, the utilization of such methods may still result in the failed isolation of a positively targeted ES cell clone. In this study, we discuss the current enhancement methods and describe an ES cell pooling strategy that maximizes the ability to detect properly targeted ES cells regardless of an inherent low targeting efficiency. The sensitivity required to detect correctly targeted events out of a pool of ES cell clones is provided by polymerase chain reaction (PCR), and only those pools containing positives need to be expanded and screened to find individually targeted clones. This method made it possible to identify targeted clones from a screen of approximately 2,300 ES cell colonies by performing only 123 PCR reactions. This technically streamlined approach bypasses the need to troubleshoot and re-engineer an existing targeting construct that is functionally suitable despite its low targeting frequency.

Fig. 1

ES cell targeting strategy. The targeting construct (a) is designed to remove exons 5, 6 and 7 of the H13 endogenous allele. (b) The targeting construct produces amplification (c–dashed line) upon homologous recombination into the locus of interest such that a neomycin designed primer comes within proximity of a 5’ flanking genomic primer. A control construct (d) simulates amplification of a positive targeting event (dashed line) even during random integration due to addition of adjacent 5’ flanking genomic sequence (bold line)

Fig. 2

Multiple electroporations are plated (a), each plate is trypsinized (b), mixed to a single cell suspension (c) and split into two fractions (d). One aliquot is analyzed by PCR, while the other is frozen for potential recovery of the positive pool. A positive scenerio is illustrated on the left, and a negative on the right. Positive clones are illustrated as filled dots

Fig. 3

Testing the sensitivity of PCR to detect a single positive ES cell colony amongst a large pool of negatives and feeder cells. One half of one positive control ES cell colony is spiked into a pool of known negatives with feeder cells (a) and is detectable by PCR (b) expected band size indicated at the left

Fig. 4

Identification of 11 positive superpools (top) and individual ES cell clones from thawed superpool #1 (bottom). Ten of 30 positive superpools are shown (top). Superpool #1 was thawed, plated and 96 colonies were screened by PCR. Three representative positives (of six total) are shown (bottom). Expected band size indicated to the left

Fig. 5

Quantitative loss of homozygosity assay. Black diamonds show the amplification efficiency (Y-axis, Ct–cycle threshold) of 3.125, 6.25, 12.5, 25 and 50 ng of wild type genomic DNA (three technical replicates each, error bars shown). Addition of 25 ng of three negative (triangles) and three positive ES cell lines (squares), illustrates that the positively targeted ES cell clones are missing one copy of exon 5. Three technical replicates each for each ES cell line are shown. X-axis is log base 10