title: Use of PCR-based methods for selection of integrated transgenes in preimplantation embryos.
abstract: The production of transgenic animals from ungulate species is an inefficient and expensive procedure. The development of selection methods to identify the small number of transgenic preimplantation embryos produced following DNA microinjection of one-cell embryos would greatly reduce both the cost and effort of these procedures. This study has examined the fate of the ovine beta-lactoglobulin-human alpha 1-antitrypsin (AATB) minigene construct or a subfragment of this following microinjection into one-cell mouse embryos. It has examined two PCR-based methods that were designed to identify a biochemical difference between microinjected DNA constructs to select preimplantation stage embryos in which chromosomal integration of exogenous DNA has occurred. The two methods involved the modification of the AATB DNA construct either by dam methylation or the substitution of dTTP by dUTP. The dam-sensitive DNA endonuclease DpnI, that was used to digest nonintegrated AATB sequences at sites located between PCR oligonucleotide sequences, was found to interfere with the activity of the subsequent PCR reaction. Analyses of the fate of dUTP-DNA indicated that either repair or replication of microinjected DNA interfered with the ability to distinguish between integrated and nonintegrated DNA constructs in the mid-preimplantation stage embryo. The distribution of microinjected AATB DNA between the blastomeres of individual four and eight-cell stage embryos was also examined by the PCR reaction. Microinjected DNA was not found to be evenly distributed between all the blastomeres of individual embryos.
authors.affiliation: Roslin Institute, Midlothian, England.
authors.forename: C
authors.initials: C
authors.lastname: Cousens
authors.forename: A S
authors.initials: AS
authors.lastname: Carver
authors.forename: I
authors.initials: I
authors.lastname: Wilmut
authors.forename: A
authors.initials: A
authors.lastname: Colman
authors.forename: I
authors.initials: I
authors.lastname: Garner
authors.forename: G T
authors.initials: GT
authors.lastname: O'Neill
mesh_terms: D000818:Animals; D001483:Base Sequence; D001755:Blastocyst / Q000502:physiology*; D001757:Blastomeres / Q000378:metabolism; D004247:DNA / Q000008:administration & dosage; D045647:DNA Glycosylases; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific; D006801:Humans; D007782:Lactoglobulins / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D012756:Sheep; D051981:Uracil-DNA Glycosidase; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D017931:DNA Primers; D007782:Lactoglobulins; D000515:alpha 1-Antitrypsin; D004247:DNA; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase