title: Increased transgene integration efficiency upon microinjection of DNA into both pronuclei of rabbit embryos.
abstract: Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.
authors.affiliation: Research Institute of Animal Production, Hlohovská 2, 949 92 Nitra, Slovak Republic. chrenekp@hotmail.com
authors.forename: Peter
authors.initials: P
authors.lastname: Chrenek
authors.forename: Dusan
authors.initials: D
authors.lastname: Vasicek
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Makarevich
authors.forename: Rastislav
authors.initials: R
authors.lastname: Jurcik
authors.forename: Karin
authors.initials: K
authors.lastname: Suvegova
authors.forename: Vladimir
authors.initials: V
authors.lastname: Parkanyi
authors.forename: Miroslav
authors.initials: M
authors.lastname: Bauer
authors.forename: Jan
authors.initials: J
authors.lastname: Rafay
authors.forename: Angelika
authors.initials: A
authors.lastname: Batorova
authors.forename: Rekha K
authors.initials: RK
authors.lastname: Paleyanda
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D004622:Embryo, Mammalian / Q000502:physiology; D005169:Factor VIII / Q000235:genetics* / Q000378:metabolism; D005260:Female; D015870:Gene Expression; D018773:Genes, erbB-1; D005821:Genetic Techniques; D016678:Genome; D049452:Green Fluorescent Proteins; D006801:Humans; D008321:Mammary Glands, Animal / Q000378:metabolism; D008845:Microinjections / Q000379:methods; D008892:Milk / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011817:Rabbits / Q000196:embryology / Q000235:genetics*; D014162:Transfection; D019076:Transgenes
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C078147:F8 protein, human; D005169:Factor VIII