title: Screening somatic cell nuclear transfer parameters for generation of transgenic cloned cattle with intragenomic integration of additional gene copies that encode bovine adipocyte-type fatty acid-binding protein (A-FABP).
abstract: Somatic cell nuclear transfer (SCNT) is frequently used to produce transgenic cloned livestock, but it is still associated with low success rates. To our knowledge, we are the first to report successful production of transgenic cattle that overexpress bovine adipocyte-type fatty acid binding proteins (A-FABPs) with the aid of SCNT. Intragenomic integration of additional A-FABP gene copies has been found to be positively correlated with the intramuscular fat content in different farm livestock species. First, we optimized the cloning parameters to produce bovine embryos integrated with A-FABP by SCNT, such as applied voltage field strength and pulse duration for electrofusion, morphology and size of donor cells, and number of donor cells passages. Then, bovine fibroblast cells from Qinchuan cattle were transfected with A-FABP and used as donor cells for SCNT. Hybrids of Simmental and Luxi local cattle were selected as the recipient females for A-FABP transgenic SCNT-derived embryos. The results showed that a field strength of 2.5 kV/cm with two 10-μs duration electrical pulses was ideal for electrofusion, and 4-6th generation circular smooth type donor cells with diameters of 15-25 μm were optimal for producing transgenic bovine embryos by SCNT, and resulted in higher fusion (80%), cleavage (73%), and blastocyst (27%) rates. In addition, we obtained two transgenic cloned calves that expressed additional bovine A-FABP gene copies, as detected by PCR-amplified cDNA sequencing. We proposed a set of optimal protocols to produce transgenic SCNT-derived cattle with intragenomic integration of ectopic A-FABP-inherited exon sequences.
authors.affiliation: College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Guo
authors.affiliation: College of Landscape Design and Forestry, Beijing University of Agriculture, Beijing, 102206, China.
authors.forename: Hejuan
authors.initials: H
authors.lastname: Li
authors.affiliation: Jiahe Hospital of Reproduction Health and Sterility, Qingdao, 266071, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: College of Life Science, Northwest University, Xi'an, 710069, China.
authors.forename: Xingrong
authors.initials: X
authors.lastname: Yan
authors.affiliation: College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China.
authors.forename: Xihui
authors.initials: X
authors.lastname: Sheng
authors.affiliation: College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China.
authors.forename: Di
authors.initials: D
authors.lastname: Chang
authors.affiliation: College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China.
authors.forename: Xiaolong
authors.initials: X
authors.lastname: Qi
authors.affiliation: College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China.
authors.forename: Xiangguo
authors.initials: X
authors.lastname: Wang
authors.affiliation: College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China.
authors.forename: Yunhai
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
authors.forename: Junya
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Animal Science and Technology, Beijing University of Agriculture, Beijing, 102206, China. nihemin@yahoo.com.cn.
authors.forename: Hemin
authors.initials: H
authors.lastname: Ni
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D030801:Animals, Genetically Modified; D002417:Cattle; D048429:Cell Size; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D005347:Fibroblasts / Q000166:cytology; D018628:Gene Dosage; D053652:Nuclear Transfer Techniques
chemical_list: D050556:Fatty Acid-Binding Proteins
keywords: A-FABP; Somatic cell nuclear transfer; Transgenic cattle