Germ-line transmission of lentiviral PGK-EGFP integrants in transgenic cattle: new perspectives for experimental embryology.
Lentiviral vectors are a powerful tool for the genetic modification of livestock species. We previously generated transgenic founder cattle with lentiviral integrants carrying enhanced green fluorescent protein (EGFP) under the control of the phosphoglycerate kinase (PGK) promoter. In this study, we investigated the transmission of LV-PGK-EGFP integrants through the female and male germ line in cattle. A transgenic founder heifer (#562, Kiki) was subjected to superovulation treatment and inseminated with semen from a non-transgenic bull. Embryos were recovered and transferred to synchronized recipient heifers, resulting in the birth of a healthy male transgenic calf expressing EGFP as detected by in vivo imaging. Semen from a transgenic founder bull (#561, Jojo) was used for in vitro fertilization (IVF) of in vitro matured (IVM) oocytes from non-transgenic cows. The rates of cleavage and development to blastocyst in vitro corresponded to 52.0 +/- 4.1 and 24.5 +/- 4.4%, respectively. Expression of EGFP was observed at blastocyst stage (day 7 after IVF) and was seen in 93.0% (281/302) of the embryos. 24 EGFP-expressing embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos, flushed from the uterus on day 15, two fetuses recovered on day 45, and a healthy male transgenic calf revealed consistent high-level expression of EGFP in all tissues investigated. Our study shows for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. The pattern of inheritance was consistent with Mendelian rules. Importantly, high fidelity expression of EGFP in embryos, fetuses, and offspring of founder #561 provides interesting tools for developmental studies in cattle, including interactions of gametes, embryos and fetuses with their maternal environment.
Bavarian Research Center for Biology of Reproduction (BFZF) e.V, Oberschleissheim, Germany. M.Reichenbach@gen.vetmed.uni-muenchen.de
Myriam
M
Reichenbach
Tiongti
T
Lim
Horst-Dieter
HD
Reichenbach
Tuna
T
Guengoer
Felix A
FA
Habermann
Marieke
M
Matthiesen
Andreas
A
Hofmann
Frank
F
Weber
Holm
H
Zerbe
Thomas
T
Grupp
Fred
F
Sinowatz
Alexander
A
Pfeifer
Eckhard
E
Wolf
D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle / Q000196:embryology / Q000235:genetics*; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D004626:Embryology / Q000379:methods*; D005260:Female; D018014:Gene Transfer Techniques; D005854:Germ Cells / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D023421:Models, Animal; D016254:Mutagenesis, Insertional; D010735:Phosphoglycerate Kinase / Q000235:genetics* / Q000378:metabolism; D011247:Pregnancy; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D019076:Transgenes / Q000235:genetics
D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D010735:Phosphoglycerate Kinase