title: Phenotypic and genotypic stability of multiple lines of transgenic pigs expressing recombinant human protein C.
abstract: The genotypic and phenotypic stability of four lines of transgenic pigs expressing recombinant human protein C in milk was examined. Two lines were established with a construct consisting of a 2.6 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Two lines were established with another construct consisting of a 4.1 kb mouse WAP promoter and a 9.4 kb human protein C genomic DNA. Genotypic stability was measured by transgene copy number transmission. Outbred offspring having a single transgene integration locus were established from a founder having three independent, multicopy loci. Phenotypic stability over multiple lactations was defined by the combination of recombinant human protein C expression levels and the isoform signature of recombinant human protein C in western blots. Both cDNA and genomic human protein C transgenes gave similar ranges of expression levels of about 100-1800 micrograms ml-1. Within a given outbred lineage having a single loci for the cDNA transgene, the expression levels ranged between 100-400 micrograms ml-1. Western blots of reduced recombinant protein C revealed that single chain content was not dependent on expression level and was consistent within each transgenic line, but varied between transgenic lines. This suggests that native swine genetics may play a role in selection of production herds with optimal post-translational proteolytic processing capability. Although swine are not conventional dairy livestock, it is agreed that the short generation times, multiple offspring per litter, stable paternal transmission of the transgene, and milk production capabilities of swine offer distinct advantages over conventional dairy livestock for the establishment of a herd producing a therapeutic recombinant protein.
authors.affiliation: Department of Chemical Engineering, Virginia Tech, Blacksburg 24061, USA.
authors.forename: K E
authors.initials: KE
authors.lastname: Van Cott
authors.forename: H
authors.initials: H
authors.lastname: Lubon
authors.forename: C G
authors.initials: CG
authors.lastname: Russell
authors.forename: S P
authors.initials: SP
authors.lastname: Butler
authors.forename: F C
authors.initials: FC
authors.lastname: Gwazdauskas
authors.forename: J
authors.initials: J
authors.lastname: Knight
authors.forename: W N
authors.initials: WN
authors.lastname: Drohan
authors.forename: W H
authors.initials: WH
authors.lastname: Velander
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006801:Humans; D007774:Lactation; D008297:Male; D008892:Milk / Q000737:chemistry / Q000378:metabolism; D008894:Milk Proteins / Q000235:genetics / Q000378:metabolism; D005810:Multigene Family; D010375:Pedigree; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011486:Protein C / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism; D013552:Swine / Q000235:genetics*; D019076:Transgenes
chemical_list: D008894:Milk Proteins; D011486:Protein C; D011994:Recombinant Proteins; C024471:whey acidic proteins