title: An approach for producing transgenic cloned cows by nuclear transfer of cells transfected with human alpha 1-antitrypsin gene.
abstract: In an attempt to produce transgenic cloned cows secreting alpha 1-antitrypsin (alpha1-AT) protein into milk, bovine cumulus cells were transfected with a plasmid containing an alpha1-AT gene and green fluorescent protein (GFP) reporter gene using Fugene 6 as a lipid carrier. The GFP-expressing cells were selected and transferred into enucleated bovine oocytes. Couplets were fused, chemically activated and cultured. Developmental competence was monitored and the number of inner cell mass (ICM) and trophectoderm (TE) cells in blastocysts were counted after differential staining. The percentage of blastocysts was lower (P < 0.05) in transgenic cloned embryos compared to non-transgenic cloned embryos (23% versus 35%). No difference in the numbers of ICM and TE cells between the two groups of embryos was observed. One or two GFP-expressing blastocysts were transferred into the uterus of each recipient cow. Out of 49 recipient cows, three pregnancies were detected by non-return estrus and rectal palpation. However, the pregnancies failed to maintain to term; two fetuses were aborted at Day 60 and 150, respectively, and one fetus at Day 240. The genomic DNA from the aborted fetus was amplified by polymerase chain reaction (PCR) to investigate integration of the transgene in the fetus. The expected PCR product was sequenced and was identical to the sequence of alpha1-AT transgene. In conclusion, the present study demonstrated that developmental competence of cloned embryos derived from transgenic donor cells was lower than embryos derived from non-transfected donor cells. Although we failed to obtain a viable transgenic cloned calf, integration of alpha1-AT gene into the fetus presents the possibility of producing transgenic cloned cows by somatic cell nuclear transfer.
authors.affiliation: Department of Therigenology and Biotechnology, College of Veterinary Medicine, Seoul National University, San56-1, Kwanak-Gu, Seoul 151-742, South Korea.
authors.forename: Goo
authors.initials: G
authors.lastname: Jang
authors.forename: M M U
authors.initials: MM
authors.lastname: Bhuiyan
authors.forename: Hyun Yong
authors.initials: HY
authors.lastname: Jeon
authors.forename: Kyeong Hee
authors.initials: KH
authors.lastname: Ko
authors.forename: Hee Jung
authors.initials: HJ
authors.lastname: Park
authors.forename: Min Kyu
authors.initials: MK
authors.lastname: Kim
authors.forename: Joung Ju
authors.initials: JJ
authors.lastname: Kim
authors.forename: Sung Keun
authors.initials: SK
authors.lastname: Kang
authors.forename: Byeong Chun
authors.initials: BC
authors.lastname: Lee
authors.forename: Woo Suk
authors.initials: WS
authors.lastname: Hwang
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D001755:Blastocyst / Q000502:physiology; D002417:Cattle / Q000235:genetics*; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000662:veterinary*; D004247:DNA / Q000032:analysis; D004624:Embryo Transfer / Q000662:veterinary; D005260:Female; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008892:Milk / Q000737:chemistry; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000254:growth & development / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011993:Recombinant Fusion Proteins; D014162:Transfection; D000515:alpha 1-Antitrypsin / Q000032:analysis / Q000235:genetics*
chemical_list: D011993:Recombinant Fusion Proteins; D000515:alpha 1-Antitrypsin; D049452:Green Fluorescent Proteins; D004247:DNA