Gene-targeted embryonic stem cells: real-time PCR assay for estimation of the number of neomycin selection cassettes

We compared the results from Southern blot, routinely used to quantify NEO copies, with the two Real-Time PCR assays. Twenty-two clones containing the single NEO copy showed values of 0.98 ± 0.24 (mean ± 2 S.D.), and were clearly distinguishable from clones with two or more NEO copies.This method was found to be useful, easy, sensitive and fast and could substitute for the widely used, but laborious Southern blot method.Two methods are available for the introduction and modification of mouse genomic DNA sequences: (i) microinjection of one or more transgenes into the pronucleus of a fertilized mouse oocyte, which usually leads to random incorporation into the genome as head-to-tail concatamers of 1-1000 units, or (ii) the use of constructs that undergo a site-specific recombination in embryonic stem cells (ES) in order to disrupt the function of a target gene (knockout) or to mutate a gene (knockin). Modified ES cells are then injected into the blastocyst [1]. In the latter case, the production of knockout or knockin ES cells is obtained through gene targeting by homologous recombination. In this work, ES cells were transfected by electroporation with a construct containing a specific genomic sequence harbouring the required mutation, along with the neomycin phophortransferase positive selection cassette (NEO) for selection of positive recombinants, flanked by two homology sequences ("arms") driving the recombination [2,3]. Homologous recombination occurs in a small number of transfected cells, resulting in the introduction of the mutation present in the targeting construct into the gene of interest. However, despite the presence of the two "arms", there may be a variable number of random integrations that may cause a position effect [4-6]. To identify the mutant ES cell clones to be microinjected, two Southern blots are usually performed: one to detect ES clones in which homologous recombination has occurred, and the other to verify the number of NEO cassettes. Usu