title: Efficient selection of preimplantation transgenic embryos by an improved procedure using Dpn I-Bal 31 digestion and the polymerase chain reaction.
abstract: Efficient selection of preimplantation transgenic embryos by an improved method after pronuclear injection of exogenous DNA is described. The method is based on subjecting DNA extracted from the embryos to restriction enzymes as well as the polymerase chain reaction (PCR). The incorporated procedure included recovery of the digested DNA with glassmilk before PCR, which markedly enhanced the rate of accurate detection of transgenic embryos. When exogenous DNA sequences in the mouse embryos were not integrated into the genome they were digested with both Dpn I and Bal 31, and subsequent PCR analysis generated DNA fragments of the injected DNA sequence in only 1.5% of cases examined. However, DNA extracted from mouse embryos containing the transgene sequences integrated into the genome evaded digestion by both enzymes and yielded transgene-specific PCR products in 68.6% of the embryos tested. When bovine embryos were used, sequences of the endogenous haemoglobin gene used as a control genomic DNA sequence were protected from enzyme digestion (PCR products in 70.5% of the embryos examined); by contrast, the non-integrated injected sequences were almost completely eliminated by the same treatment (PCR products in 1.4% of the embryos examined). It is suggested that this method might be useful for the selection of transgenic embryos before embryo transfer, thereby reducing the number of recipient females required.
authors.affiliation: Department of Applied Genetics, University of Tokyo, Japan.
authors.forename: B B
authors.initials: BB
authors.lastname: Seo
authors.forename: C H
authors.initials: CH
authors.lastname: Kim
authors.forename: H
authors.initials: H
authors.lastname: Tojo
authors.forename: S
authors.initials: S
authors.lastname: Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Yamanouchi
authors.forename: M
authors.initials: M
authors.lastname: Takahashi
authors.forename: T
authors.initials: T
authors.lastname: Sawasaki
authors.forename: C
authors.initials: C
authors.lastname: Tachi
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000737:chemistry*; D004247:DNA / Q000032:analysis*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D047108:Embryonic Development; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D005260:Female; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D019076:Transgenes
chemical_list: D004247:DNA; D004706:Endodeoxyribonucleases; C038407:exonuclease Bal 31; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific