EXPRESSION OF HUMAN BLOOD-CLOTTING FACTOR-VIII (FVIII) CONSTRUCTS IN THE MAMMARY-GLAND OF TRANSGENIC MICE AND SHEEP

The aim of this study is to produce transgenic mice and sheep which ex press large amounts of human anti-hemophilic factor VIII (FVIII) in th e mammary gland. To overcome the potentially low expression of cDNA-co nstructs we have added heterologous introns from the murine metallothi onein (MT-I) gene, resulting in gene constructs of 13 200 and 14 400 b p, respectively. Via microinjection of zygotes recovered from superovu lated donors, we have produced 25 lines of transgenic mice with four d ifferent constructs of which two are under the control of the beta-lac toglobulin (beta-Lac) promoter and two under the control of the Whey a cidic protein (WAP) promotor. One of the beta-Lac- or WAP-hFVIII const ructs possessed the MT-I fragment containing both introns in the 3'-un translated region of the FVIII cDNA, respectively. Expression of PVIII cDNA was determined in mammary tissue of the transgenic mouse lines b y reverse transcriptase PCR (RT-PCR). Of these 25 transgenic lines 13 (52%) expressed the integrated gene construct (WAP hFVIII 7/4, WAP hFV III MT-I 11/5, beta-Lac hFVIII 2/1, beta-Lac hFVIII MT-I 5/3). By rest riction enzyme analysis of the PCR products and Southern Blot analysis with FVIII probes we confirmed specificity of the expression of the t ransgene. Following microinjection of beta-Lac hFVIII or beta-Lac hFVI II MT-I constructs we have generated 8 transgenic founder sheep. One b eta-Lac hFVIII MT-I sheep expressed FVIII in the Iactating mammary gla nd as detected by RT-PCR from biopsied mammary gland tissue. Two male founder animals transmitted the transgene in a Mendelian fashion to th eir offspring. To achieve site independent expression, new gene constr ucts employing matrix-attachment region elements (MAR) (MAR-beta-Lac-h FVIII MT-I) were recently microinjected into pronuclei and two female and two male founder lambs were obtained. The total efficiency of micr oinjection into pronuclei of ovine zygotes has been 2.9% transgenic la mbs (12/413). Analysis of mouse and sheep milk using two different clo tting assays and a sandwich ELISA did not reliably indicate the presen ce of active FVIII in milk. Currently, identification of FVIII in the milk of transgenic founder and F-1 females with the aid of a sensitive antibody is under investigation.