title: Detection of microinjected genes in bovine preimplantation embryos with combined DNA digestion and polymerase chain reaction.
abstract: We have developed a simple digestion-polymerase chain reaction (PCR) assay for a simultaneous transgene detection and sexing of pronucleus-injected bovine preimplantation embryos. Bovine embryos were microinjected with dam-methylated gene construct and cultured in vitro for 6-7 days after the injections. The developed blastocysts and compact morulae were bisected and the embryonic biopsies representing mainly trophoblasts were subjected to the digestion-PCR, while the biopsied embryos remained in culture. Embryonic DNA was released with proteinase K and the samples were digested with a Dpnl-Bal31 mixture before the PCR amplification of the transgene, bovine alpha S1-casein, and bovine Y-chromosome fragments in the same reaction. The whole assay from biopsy to electrophoresis took less than 6 hr. The digestion removed up to 50 fg of dam-methylated transgene copies (unintegrated or contaminants) and also a few hundred copies of contaminating PCR products from the embryonic samples. The digestion-PCR assay eliminated all transgene contaminations from noninjected blastocysts, which were exposed to the microinjection DNA during the stay in injection chambers, and reduced the amount of transgene-positive embryos among pronucleus-injected blastocysts as compared with unmodified PCR. Analysis of 486 microinjected bovine embryo biopsies in 13 separate experiments revealed that we were able to sex 398 (82%) of the biopsies and 77 (19%) of the biopsies were scored as transgene positive and 57 (14%) as transgene questionable. Upon reanalysis of 41 of the biopsied embryos, 38 (93%) of the embryos were observed to be transgene negative and 2 questionable in both assays and uneven distribution of transgene copies was observed in one embryo. The results from sexing were in accordance with biopsies and remaining embryos in 38 (93%) of the embryos.
authors.affiliation: Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
authors.forename: J M
authors.initials: JM
authors.lastname: Hyttinen
authors.forename: T
authors.initials: T
authors.lastname: Peura
authors.forename: M
authors.initials: M
authors.lastname: Tolvanen
authors.forename: J
authors.initials: J
authors.lastname: Aalto
authors.forename: J
authors.initials: J
authors.lastname: Jänne
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst; D002417:Cattle; D005260:Female; D018014:Gene Transfer Techniques; D016133:Polymerase Chain Reaction / Q000379:methods; D011247:Pregnancy; D012732:Sex Determination Analysis / Q000379:methods