44 POSSIBILITY OF PRODUCTION OF A TRANSGENIC CLONED COW USING THE SOMATIC CELL NUCLEAR TRANSFER OF HUMAN ALPHA 1-ANTITRYPSIN GENE-TRANSFECTED CELLS
G. Jang A , M.M.U. Bhuiyan A , E.S. Park B , H.Y. Chun A , J.G. Cho C , H.J. Park A , S.K. Kang A , B.C. Lee A and W.S. Hwang AA Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Seoul National University, Seoul, South Korea. email: firstlee@snu.ac.kr;
B School of Agricultural Biotechnology, Seoul National University;
C College of Veterinary Medicine, Chungnam National University, Daejeon, South Korea.
Reproduction, Fertility and Development 16(2) 144-144 https://doi.org/10.1071/RDv16n1Ab44
Submitted: 1 August 2003 Accepted: 1 October 2003 Published: 2 January 2004
Abstract
The present study was designed to produce, using a somatic cell nuclear transfer (SCNT), transgenic cloned cows that secreted a therapeutic protein into milk. Bovine cumulus cells were transfected with a plasmid that contained the alpha 1-antytrypsin DNA and green fluorescent protein (GFP) reporter gene using a Fugene 6 as a lipid carrier. In vitro-matured bovine oocytes were enucleated followed by confirmation of enucleation via nuclear staining. The GFP-expressing donor cells with the transgene were selected under blue fluorescent light just before injection of cells. The SCNT was performed according to the established system in our laboratory (Jang G et al., 2003 Reprod. Fertil. Dev. 15, 179–185). The couplets were fused, chemically activated, and cultured in modified synthetic oviduct fluid (mSOF) at 39°C in a humidified atmosphere of 5% CO2 in air for up to 7 days. The developmental ability (in Experiment 1) and number of inner cell mass (ICM) and trophectoderm (TE) cells (in Experiment 2) in transgenic and non-transgenic cloned embryos determined by differential staining as described by Thouas et al., (2001 Reprod. Biomed Online 3, 35–29) were compared. In Experiment 3, only GFP-expressing blastocysts were transferred to the uteri of recipient cows. A general linear model consisting of ANOVA and a least squares method in the SAS program was used for statical analysis. The percentage of blastocysts was lower (P < 0.05) in transgenic cloned embryos compared to non-transgenic embryos (34% v. 20%). No difference (P < 0.05) in the cell number of ICM and TE cells between two groups of embryos was observed. Out of 47 recipient cows, three pregnancies were detected by non-return to estrus and by rectal palpation. However, the pregnancies were not maintained to term; 2 fetuses were aborted at Days 60, and 150, and one fetus was aborted at Day 240. The genomic DNA from the fetus aborted at Day 240 was prepared and analyzed for the expression of GFP and alpha 1-antitrypsin gene by polymerase chain reaction. The expected PCR products (710 bp for alpha 1-antytrypsin DNA and 431 bp for GFP) were obtained and confirmed by sequence analysis. In conclusion, the present study demonstrated that developmental competence of cloned embryos derived from transgenic donor cells was lower compared with cloned embryos derived from non-transfected donor cells. Although we failed to produce a viable transgenic cloned calf, expression of alpha 1-antitrypsin in fetal tissue represented the possibility of production of a transgenic cloned cow by SCNT. This study was supported by Biogreen 21-1000520030100000.
