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United States Department of Agriculture

Agricultural Research Service

Title: Expression of Gene-Gun Injected Plasmid DNA in the Ovine Mammary Gland and in Lymph Nodes Draining the Injection Site.

Authors
item Kerr, David - ALTRABIO, INC.
item Furth, Priscilla - U OF MD MEDICAL SCHOOL
item Powell, Anne
item Wall, Robert

Submitted to: Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 13, 1995
Publication Date: N/A

Interpretive Summary: Genetic enginerring has the potential of rapidly improving genetic characteristics of livestock. However the technology is limited by cumbersome, and sometimes unreliable procedures for testing new genetic strategies. We are developing a means to overcome that limitation by "shooting" newly designed genes into animals. We have previously shown that genes introduced in that manner are taken up by cells in the living animal. This study was designed to determine if those genes function normally. Our "target" was the mammary gland of lactating sheep. A gene-gun, which delivers a DNA solution as a jet stream with sufficient force to pentrate 3 to 5 cm into the mammary gland, was used to introduce a gene encoding human growth hormone (hGH). We discovered that the gene functioned properly, producing both mRNA and hGH and small quantities of hGH protein in the milk. This technique will now enable us to study the genes involved in milk production. We also detected serum antibodies to the hGH demonstrating the potential of this gene-gun technique as a vaccination strategy. Studies are now underway to develop a DNA based vaccine for Cryptosporidiosis, and to produce pharmaceutical proteins, such as human blood clotting factors, in the sheep milk which could then be purified for use in humans.

Technical Abstract: We have further evaluated a jet-injection based DNA delivery system as a means to transiently transfect the lactating mammary gland in vivo and as a technique for DNA vaccination. The model expression plasmid contained the human growth hormone (hGH) structural gene driven by the human cytomegalovirus immediate early gene 1 promoter/enhancer region (CMV). Expression from plasmid DNA jet-injected into lactating mammary glands of sheep was sufficient to be detected by Northern blod analysis when tissue was obtained 48 hours after in vivo transfection. In contrast, following DNA transfer by needle and syringe RNA was detectable by RT-PCR, but not by Northern blot analysis. Furthermore, specific mRNA was detected by RT-PCR in lymph nodes draininig the mammary gland injection sites. In a second experiment, the time course and magnitude of antibody development to hGH following jet-injection of CMV-hGH, into either muscle or lactating mammary tissue in vivo circumvents the difficulties encountered with in vitro culture techniques and provides the potential for examining mammary reglatory elements and testing of fusion gene constructs designed for the production of transgenic large animal bioreactors.

Last Modified: 4/15/2014
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