432 INHERITANCE OF LENTIVIRAL PHOSPHOGLYCERATE KINASE-ENHANCED GREEN FLUORESCENT PROTEIN (PGK-eGFP) INTEGRANTS OF TRANSGENIC CATTLE
M. Reichenbach A , F. A. Habermann C , H. D. Reichenbach F , T. Guengoer B , F. Weber G , H. Zerbe G , F. Sinowatz C , A. Pfeifer D and E. Wolf BA Bavarian Research Center for Biology of Reproduction (BFZF) e.V., Oberschleissheim, Germany;
B Chair for Molecular Animal Breeding and Biotechnology, LMU Munich, Munich, Germany;
C Chair for Veterinary Anatomy, Histology and Embryology, LMU Munich, Munich, Germany;
D Institute of Pharmacology and Toxicology, University of Bonn, Bonn, Germany;
E Pharma Center Bonn, University of Bonn, Bonn, Germany;
F Institute for Animal Breeding, Bavarian State Research Center for Agriculture, Grub, Germany;
G Clinic for Ruminants, Center of Clinical Veterinary Medicine, LMU Munich, Oberschleissheim, Germany;
H Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Munich, Germany
Reproduction, Fertility and Development 22(1) 373-373 https://doi.org/10.1071/RDv22n1Ab432
Published: 8 December 2009
Abstract
An alternative approach to classic techniques for the generation of transgenic livestock is the use of viral vectors. Using lentiviral vectors (LV) we previously generated transgenic founder cattle with integrants carrying phosphoglycerate kinase (PGK) promoter-enhanced green fluorescent protein (eGFP) expression cassettes (Hofmann et al. 2004 Biol. Reprod. 71, 405-409). The aim of this work was to investigate the transmission of LV-PGK-eGFP integrants through the female and male germ line of transgenic founder cattle in resulting embryos, fetuses, and offspring. The female founder animal was superovulated and artificially inseminated with a nontransgenic bull. Six of the 16 embryos obtained were transferred to synchronized recipient heifers, resulting in 2 pregnancies and birth of 1 healthy male transgenic calf, expressing eGFP as detected by in vivo imaging and real-time PCR. Cryopreserved semen of the founder bull and matured COC of nontransgenic cows were used for in vitro embryo production as previously described by Hiendleder et al. (2004 Biol. Reprod. 71, 217-223). The rates of cleavage and development to blastocysts in vitro corresponded to 52.3 ± 3.8% and 23.5 ± 4.6%, respectively. In vivo expression of eGFP was observed at blastocyst stage (Day 7 after IVF) and was seen in 93.8% (198/211) of all blastocysts. Twenty-four eGFP-positive embryos were transferred to 9 synchronized recipients. Analysis of 2 embryos flushed on Day 15, 2 fetuses recovered on Day 45, and a healthy male transgenic calf revealed consistent high-level expression of eGFP in all tissues investigated. These observations show for the first time transmission of lentiviral integrants through the germ line of female and male transgenic founder cattle. Although eGFP transgenic cattle have been produced before by nuclear transfer from transfected cells, lentiviral transgenesis has the advantage that only one copy of the provirus is integrated at a particular chromosomal integration site. High-fidelity expression of eGFP in embryos, fetuses, and offspring of founders provides an interesting tool for developmental studies in cattle, including interactions of gametes, embryos, and fetuses with their maternal environment.
