Development of a recombinant bovine leukemia virus vector for delivery of a synthetic bovine growth hormone-releasing factor gene into bovine cells.
Continuous intravenous infusion of bovine growth hormone-releasing factor (bGRF) increases milk synthesis in dairy cattle by as much as 46%. We have begun to develop a system for delivery and expression of a synthetic bGRF gene in cultured bovine cells using the provirus of the bovine leukemia virus (BLV). The gene encoding synthetic bGRF, constructed from eight overlapping oligonucleotides, was fused to the whey acidic protein promoter (WAP) or the mouse mammary tumor virus promoter (MMTV). These plasmids, termed pWAP.GRF and pMMTV.GRF, were able to induce transcription of bGRF upon transfection into Madin-Darby bovine kidney (MDBK) cells and induction with a lactogenic hormonal milieu (prolactin, hydrocortisone, triiodothyronine, insulin) or dexamethasone. When these constructs were cloned into a BLV vector in place of its oncogenic region, and transfected into MDBK cells, bGRF was expressed. Virus particles were prepared from these cultures and used to deliver the bGRF gene by viral infection into fresh MDBK cells. Northern blot analysis of MDBK total RNA revealed a fivefold higher level of expression of bGRF mRNA in transfected cultures than in virally infected cells, and no expression was detected in control cultures. The bGRF peptide was detected in both cell extracts and media samples from transfected cultures but was not detected in cell extracts or media samples from virally infected cells. This provirus construct may prove useful as a delivery system for peptides into cattle.
Mehigh
C S
CS
Department of Animal Science, Michigan State University, East Lansing 48824.
Elias
V D
VD
Mehigh
R J
RJ
Helferich
W G
WG
Tucker
H A
HA
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D016355:Genes, pX; D005822:Genetic Vectors; D013007:Growth Hormone-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics*; D015151:Immunoblotting; D001909:Leukemia Virus, Bovine; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D014158:Transcription, Genetic; D014162:Transfection
D012333:RNA, Messenger; D013007:Growth Hormone-Releasing Hormone