Knockout of one acetylcholinesterase allele in the mouse

One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) c ells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1 .6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this v ector deleted exons 2-5, which removed 93% of the ACHE coding sequence incl uding the signal peptide, the active site serine, and the histidine and glu tamic acid of the catalytic triad. The gene targeting vector was transfecte d into ES cells by electroporation. Colonies resistant to G418 and gancyclo vir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for micr oinjection into C57B1/6 mouse blastocysts. The injected blastocysts were im planted into pseudopregnant CD/1 white mice. More than 200 injected blastoc ysts were transferred into 20 mice. More than 65 mice were born, of which 1 1 were chimeras. Chimeras were identified by their black and agouti coat co lor. Littermates were all black. Thus far, seven male chimeras have been br ed with more than 130 C57B1/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contribut ed to the germline. Offspring with agouti coat color have a 50% chance of c arrying the knockout allele. The 26 agouti offspring were screened for an A CHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are hetero zygous ACHE knockout mice, and they are healthy and alive at 29 days of age . We expect a phenotype to appear in nullizygous animals. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.