Production of transgenic sheep: Microinjection of the bovine P77 gene construct in Chios sheep breed zygotes


The feasibility of integration of the bovine P77 construct into zygotes and two cell embryos of Chios sheep breed was examined. The P77 gene construct comprised bovine as l casein promoter regulatory sequence fused to the fragment between exons 2 and 9 of the bovine chymosin gene. One of the two pronuclei of the zygote or a nucleus of a blastomere of a two cell embryo was microinjected with 1 pi of DNA solution containing approximately 1000 copies of the gene construct. In total 193 zygotes and 87 two cell embryos collected from 49 donor ewes were used. After 1 - 5 h culture microinjected zygotes (n=175) and two cell embryos (n=76) were transferred by a laparoscopic technique to the oviducts of 68 recipients. 22 recipients (32.4%) gave birth to 29 lambs which correspond to 11.6% of the total number of zygotes - embryos transferred. PCR analysis performed in skin samples of these lambs revealed that in one ewe lamb the P77 construct has been integrated in its genome. The growth and puberty of this lamb was physiological and is currently pregnant. This is the first transgenic farm animal born in Greece.

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Rucker ΕΒ, Piedrahita JA. Cre-mediated recompination at the murine whey acidic protein (mWAP) locus. Theriogenology 1997,47: 228
Lee CS, Choi YH, Oh KB, Kang YK, Lee KK. Temporal – and spatial specific expression of bovine b-casein/bovine growth hormone fusion gene in transgenic mice. Theriogenology 1997,47:225
Gavin WG, Pollock D, Fell P, Yelton D, Cammuso C, Harrington M, Lewis-Williams J, Midura P, Oliver A, Smith TE, Wilburn B, Echelard Y, Meade H. Expression of the antibody hBR96-2 in the milk of transgenic mice and production of hBR96-2 transgenic goats. Theriogenology 1997,47:214.
Hammer RE, Pursel VG, Rexroad CE, Wall RJ, Bolt DJ, Ebert KM, Palmiter RD, Brinster RL. Production of transgenic Rabbits, Sheep and Pigs by microinjections. Nature 1985,315:680-683.
Rexroad CE, Hammer RE, Behringer RR, Palmiter RD, Brinster RL. Insertion expression and physiology of growth vepulating genes in ruminants J Reprod Fert 1990, (supp.) 41:119-124
Rexroad CE, Mayo K, Bolt DJ, Elsasser ΤΗ, Miller KF, Berrtinger RR, Palmiter RD, Brinster RL. Transferrin and albumin directed expression of growth - related peptides in transgenic sheep. J anim Sci 1991, 69 (7) : 2995-3004.
Murray JD, Nancarow CD, Marshall JT, Hazelton IG, Ward KA. Production of transgenic merino sheep by microinjection of ovine metallotheionine - ovine growth hormone fusion genes. Reprod Fertil Develop 1989,1:147-155.
Ward KA, Nancarrow CD. The genetic engineering of production traits in domestic animals. Experimentia 1991,47: 913-922.
Powell BC, Walker SK, Bawden CS, Sivaprasad AV, Rogers GE. Transgenic sheep and wool growth : possibilities and current status. Reprod Fertil Develop 1994, 6(5) : 615-623.
Damak S, Jay NP, Barell GK, Bullock DW. Targeting gene expression to the wool follicle in transgenic sheep. Biotechnology, 1996,14 (2): 181-184.
Damak S, Su-Hung Y, Jay NP, Bullock DW, Su HY. Improved wool production in transgenic sheep expressing insulin like growth factor 1. Biotechnology, 1996,14(2):185-188.
Lo D, Pursel V, Linton PJ, Sandgren E, Behringer R, Rexroad C, Palmiter RD, Brinster RL. Expression of mouse IgA by transgenic mice pigs and sheep. Eur J Immunol 1991, 21:1001-1006.
Clements JE, Wall RJ, Narayan O, Hauer D, Sheffer D, Powell AM, Zink MC, Rexroad CE. Transgenic sheep that express the visna virus envelope gene. Theriogenology 1994, 41:180.
Simons JP, Wilmut I, Clark AJ, Archibald AL, Bishop JO, Lathe R. Gene transfer into sheep. Biotechnology 1988, 6: 179-183.
Carver A, Wright G, Cottom D, Cooper J, Dalrymple M, Temperley S. Udell M, Reeves D, Percy J, Scott A, Barrass D, Gibson Y, Jeffrey Y, Samuel C, Colman A, Garner I. Expression of human alpha 1 antitrypsin in transgenic sheep. Cytotechnology 1992 9: 77-84.
McClenaghan M, Archibald AL, Harris S, Simons JP, Writelaw CBA, Wilmut I, Clark AJ. Production of human alpha 1 - antitrypsin in the milk of transgenic sheep and mice: targeting expression of cDNA sequences to the mammary gland. Animal Biotechnology 1991,2:161-176.
Clark AJ, Bessos H, Bishop JO, Brown P, Harris S, Lathe R, McClenaghan M, Prowse C, Simons JP, Whitelaw CBA, Wilmut I. Expression of human anti-hemophilic factor IX in the milk of transgenic sheep. Biotechnology 1989,7:487-492.
Schnieke AE, Kind AJ, Ritchie WA, Mycock K, Scott AR, Ritchie M, Wilmut I, Colman A, Campbell KHS. Human factor IX transgenic sheep produced by transfer of nuclei from ttansfected fetal fibroblasts. Science, Washington, 1997, 278 (5346) : 2130-2133.
Halter R, Carnwath J, Espanion G, Herrmann D, Lemme E, Niemann Η, Paul D. Strategies to express factor VIII gene constructs in the ovine mammary gland. Theriogenology 1993,39:137-149.
Brem G, Hartl P. High-level expression of prochymosin in the milk of transgenic rabbits. In Frontiers of Biotechnology in Agriculture, Conference 1-4 August, 1991, Sea of Galilee, Israel
Brem G, Besenfelder U, Zinovieva N, Seregi J, Solti L, Hartl P. Mammary gland specific expression of chymosin constructs in transgenic Rabbits. Theriogenology 1995,43(1 ) :175.
Ernst LK, Gol'dman IL, Zivov'-eva NA, Bashkeev ED, Gogolevskii PA, Kadulin SG, Brem G. Production of transgenic sheep carrying the gene construction alpha SI casein/chymosin Doklady - Biochemistry 1995,345(4):187-189.
Boscos C, Vainas E, Kouskoura Th, Samartzi F, Vafiadis D, Dellis S. Superovulatory response of Chios and Friesian ewes to two FSH-P dose levels. Reprod dorn anim. 1997 (32):195-198.
Kuehholzer B, Endoskopische Embryotransferverfahren bein Schaf. Wien vet. med. U, Diss. 1996.
Hidaka M, Sasaki K, Unzumi Τ, Beppu T. Cloning and structural analysis of the calf prochymosin gene. Gene 1986, 43:197-203
Bonsing J, Mackinlay AG. Recent studies on nucleotide sequences encoding the caseins. J Dairy Res 1987,54:447-461
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K. Current protocols in molecular biology John Willey and Sons, 1987, New York.
Colman A. Production of proteins in the milk of transgenic livestock : problems, solutions and successes. Am J Clin Nutr. 1996, 63 (4), 639-645.
Menck MC, Mercier Y, Lobo RB, Heyman Y, Renard JP, Thompson EM. In vivo luminescent selection of putative transegenic bovine blastocysts. Theriogenology 1997,47:227.
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