title: Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.
abstract: Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine β-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.
authors.affiliation: Departamento de Ciências Básicas, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brasil monzani.paulo@gmail.com.
authors.forename: P S
authors.initials: PS
authors.lastname: Monzani
authors.forename: J R
authors.initials: JR
authors.lastname: Sangalli
authors.forename: T H C
authors.initials: TH
authors.lastname: De Bem
authors.forename: F F
authors.initials: FF
authors.lastname: Bressan
authors.forename: P
authors.initials: P
authors.lastname: Fantinato-Neto
authors.forename: J R V
authors.initials: JR
authors.lastname: Pimentel
authors.forename: E H
authors.initials: EH
authors.lastname: Birgel-Junior
authors.forename: A M
authors.initials: AM
authors.lastname: Fontes
authors.forename: D T
authors.initials: DT
authors.lastname: Covas
authors.forename: F V
authors.initials: FV
authors.lastname: Meirelles
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001947:Breeding / Q000379:methods*; D002364:Caseins / Q000235:genetics; D002417:Cattle / Q000235:genetics*; D002874:Chromosome Mapping; D019976:Cloning, Organism; D053938:DNA Fragmentation; D004622:Embryo, Mammalian / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D005164:Factor IX / Q000096:biosynthesis* / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D053652:Nuclear Transfer Techniques; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D002364:Caseins; D011994:Recombinant Proteins; D005164:Factor IX