Reproductive wastage is common in women, and greatly reduces the efficiency of livestock production; most of this loss is due to embryonic mortality from faulty maternal recognition of pregnancy. Proteins secreted by the concept us that communicate its presence to the mother have been identified: in sheep, ovine trophoblast protein-1 (oTP-1); cattle, (bTP- 1); and humans, human chorionic gonadotropin (hCG). Furthermore, administration of exogenous hCG to cattle and sheep in early pregnancy decreases embryonic death. Our strategy is to enhance mechanisms of maternal recognition of pregnancy in transgenic sheep by: (1) expressing hCG in ovine trophoblast; and (2) altering the temporal pattern of oTP-1 expression. Problems besetting previous research on transgenic sheep, namely inappropriate transgene expression and long gestation intervals, will be circumvented as follows: 1)Transgene expression will be limited to trophoblast and thus should not interfere with the normal physiology of the embryo, lamb, or adult. 2)Before making transgenic animals, recombinant DNA constructs will be transfected into primary cultures of trophoblast cells from day-15 and day- 24 conceptuses, and only promising constructs will be used to make transgenic animals. 3)Only transgenic embryos will be transferred; transgene status will be determined from DNA of a few biopsied cells, amplified by the polymerase chain reaction. 4)Embryos will be bisected and demi-embryos transferred, permitting rapid analysis of the transgenic phenotype of one twin at 16 days of gestation, while the identical twin develops to term in another recipient.
Specific aims are: 1)Determine if the 5' flanking region of either oTP-1 or bTP-1 is able to direct stage-specific expression of a heterologous gene (luciferase) in ovine trophoblast in primary culture. 2)Determine if biologically active hCG can be produced in cultured ovine trophoblast using regulatory sequences characterized in specific aim 1. 3)Determine if cultured sheep trophoblast can express the hCG beta subunit gene under control of its native regulatory region. 4)If (3) above is affirmative, determine if the regulatory region of the hCG beta gene can be used to direct expression of oTP-1 and/or biologically active hCG in cultured ovine trophoblast. If (3) is negative, determine if the regulatory region of the hCG alpha gene can express hCG alpha and beta subunits in ovine trophoblast. 5)Make transgenic sheep with two promising constructs from the above studies.
| Glover, Michelle D; Seidel Jr, George E (2003) Increased messenger RNA for allograft inflammatory factor-1, LERK-5, and a novel gene in 17.5-day relative to 15.5-day bovine embryos. Biol Reprod 69:1002-12 |
| Keller, M L; Roberts, A J; Seidel Jr, G E (1998) Characterization of insulin-like growth factor-binding proteins in the uterus and conceptus during early conceptus elongation in cattle. Biol Reprod 59:632-42 |
