Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene

Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro. The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques. Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene. The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells. To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed. The neo gene was detected in approximately half of the embryos. The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development.     

Localization of a cis-acting element responsible for the developmentally regulated amplification of Drosophila chorion genes

Late in oogenesis two clusters of Drosophila chorion genes and flanking DNA sequences undergo specific amplification in ovarian follicle cells. Lines were constructed using P-element-mediated transformation in which DNA segments derived from the chorion gene cluster at 66D on chromosome III had been inserted at new chromosomal locations. Only transposons that contained a specific 3.8 kb genomic segment derived from the cluster underwent amplification during oogenesis, which occurred with apparently normal tissue and temporal specificity. Adjacent nonchorion sequences also underwent amplification. However, the ability of a transposon to replicate differentially was subject to position effect. These studies provide evidence for the existence of a specific, cis-acting element controlling chorion gene amplification, which includes an origin for disproportionate DNA replication. Attempts to induce amplification with subfragments of the 3.8 kb segment were unsuccessful, suggesting that much of this fragment may be required for amplification.