title: Influence of time of gene microinjection on development and DNA detection frequency in bovine embryos.
abstract: The effect of DNA microinjection at various times after in vitro insemination on DNA detection and survival rates of bovine embryos was investigated. Oocytes were inseminated 24 h after maturation with frozen/thawed semen prepared with a Percoll separation procedure. At 11, 15 and 19 h after insemination, embryos were centrifuged to visualize pronuclei and microinjected with a murine whey acidic protein-human protein C genomic DNA construct. After culture for 7 days on Buffalo Rat Liver cells, embryos were assessed for stage of development and assayed for the presence of the transgene by polymerase chain reaction. Of zygotes in the 11 h after insemination treatment, 16% (25/152) of non-injected and 7% (11/161) of injected embryos developed to the morula or blastocyst stage. Comparable development of non-injected and injected embryos treated at 15 h after insemination was 15% (23/158) and 4% (6/159) and treated at 19 h after insemination was 14% (23/162) and 1% (1/165), respectively. Development of injected embryos was greater (p < 0.05) when injection was performed at 11 h after insemination compared to 19 h after insemination. Development of non-injected embryos was greater (p < 0.01) than that of injected embryos. There was no difference in transgene detection frequency in embryos of all developmental states between treatments (53% at 11; 50% at 15; 48% at 19 h after insemination). Injected embryos testing positive for the presence of the transgene exhibited increased development over negative embryos (p < 0.01). Greater development efficiencies can be obtained in microinjected bovine embryos when injection is performed early in pronuclear formation.
authors.affiliation: Department of Dairy Science, Virginia Polytechnic Institute and State University, Blacksburg 24061-0315.
authors.forename: R L
authors.initials: RL
authors.lastname: Krisher
authors.forename: J R
authors.initials: JR
authors.lastname: Gibbons
authors.forename: R S
authors.initials: RS
authors.lastname: Canseco
authors.forename: J L
authors.initials: JL
authors.lastname: Johnson
authors.forename: C G
authors.initials: CG
authors.lastname: Russell
authors.forename: D R
authors.initials: DR
authors.lastname: Notter
authors.forename: W H
authors.initials: WH
authors.lastname: Velander
authors.forename: F C
authors.initials: FC
authors.lastname: Gwazdauskas
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology / Q000235:genetics*; D001483:Base Sequence; D002417:Cattle; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004624:Embryo Transfer; D005314:Embryonic and Fetal Development / Q000235:genetics*; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D051379:Mice; D008845:Microinjections; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011486:Protein C / Q000235:genetics; D013997:Time Factors
chemical_list: D017931:DNA Primers; D011486:Protein C; D004247:DNA