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NON TECHNICAL SUMMARY:
Approximately 30 percent of dairy cows will experience a course of mastitis during their annual lactation cycle. The estimated cost of this disease to the US dairy industry is two billion dollars per year. The long-term goal of our research is to develop dairy cattle with enhanced resistance to microbial infection of the mammary gland. Our approach is to use transgenic animal technology to enable mammary cells to produce additional anti-bacterial enzymes. Transgenic cattle are very expensive to generate and thus proposed new genes are tested in transgenic mice. We have generated transgenic mice that contain the bacterial gene for lysostaphin, a potent antibacterial enzyme. This gene is under the control of a milk protein regulatory region such that lysostaphin is only produced by lactating mammary cells. These mice secrete lysostaphin into their milk and have markedly enhanced resistance to staphylococcal mastitis. The first objective of the current proposal is to
document the effects, if any, of lysostaphin on the production, composition, and nutritional quality of milk produced by these mice. Our second objective is to examine a new gene regulatory unit that appears to be relatively silent during normal lactation but will potentially direct lysostaphin production in response to infection and during the mastitis-prone time periods shortly before and immediately following a lactation period. The regulatory unit will be isolated from the bovine genome and will be used for the generation of new transgenic mice that will then be evaluated for mastitis resistance.
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| APPROACH:
Transgenic mice that secrete lysostaphin into their milk have been produced. Additional characterization will be performed. This characterization includes an evaluation of more milk samples for lysostaphin content and activity, total protein, and protein profiles (PAGE). Northern blot analysis of lactating mammary tissue for lysostaphin and other milk proteins (whey acidic protein, b-casein) will be performed as will tissue distribution of lysostaphin mRNA. To assess milk yield a weigh-suckle-weigh experiment comparing the performance of non-transgenic controls and lysostaphin expressing transgenic mice will be conducted. A second weigh-suckle-weigh experiment will compare the growth of non-transgenic pups suckling transgenic or non-transgenic mice. For objective two, a bovine regulatory region that is induced by infection and involution will be cloned from a bovine genomic library. The regulatory region will be ligated to the modified lysostaphin gene and the
resulting construct used to generate transgenic mice. Milk will be obtained from these mice before and 24 hours after pup removal to assess the ability of the regulatory region to direct expression of lysostaphin following involution. In a similar fashion, milk will be obtained before and 24 hours after a bacterial challenge to assess the infection responsiveness of the regulatory region.
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CRIS NUMBER: 0186427
SUBFILE: CRIS
PROJECT NUMBER: VT-AS-034CG
SPONSOR AGENCY: CSREES
PROJECT TYPE: NRI COMPETITIVE GRANT
PROJECT STATUS: TERMINATED
MULTI-STATE PROJECT NUMBER: (N/A)
START DATE: Dec 1, 2000
TERMINATION DATE: Nov 30, 2004
GRANT PROGRAM: IDENTIFYING GENETIC MECHANISMS & GENE MAPPING
GRANT PROGRAM AREA: Animal Systems
CLASSIFICATION
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| 303 | 3410 | 1030 | 2.2 | 10% |
| 303 | 3410 | 1040 | 2.2 | 20% |
| 303 | 4010 | 1040 | 2.2 | 10% |
| 311 | 3410 | 1040 | 4.2 | 40% |
| 311 | 4010 | 1030 | 4.2 | 10% |
| 311 | 4010 | 1040 | 4.2 | 10% |
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CLASSIFICATION HEADINGS
KA311 - Animal Diseases
KA303 - Genetic Improvement of Animals
S4010 - Bacteria
S3410 - Dairy cattle, live animal
F1030 - Cellular biology
F1040 - Molecular biology
G2.2 - Increase Efficiency of Production and Marketing Systems
G4.2 - Reduce Number and Severity of Pest and Disease Outbreaks
RESEARCH EFFORT CATEGORIES
| BASIC |
50% |
| APPLIED |
50% |
| DEVELOPMENTAL |
(N/A)% |
KEYWORDS: mastitis; transgenic animals; mice; anti bacterial agents; infection; disease prevention; involution; milk production; dairy cattle; cell biology; animal genetics; molecular biology; bacterial diseases (animals); animal diseases; animal health; gene expression; milk yields; milk quality; gene regulation; milk protein; protein content
PROGRESS: Dec 1, 2000 TO Nov 30, 2004
The lines of transgenic mice were generated that produce an anti-staphylococcal enzyme (lysostaphin) into their milk. The lines were designated low, medium, and high expression based on the amounts of lysostaphin produced. Milk for mice within low, medium, and high-expressing lines contains approximately 0.06, 0.13, or 1.0 mg/ml of the lysostaphin variant. Lactating mice were challenged with an intramammary infusion of 104 cfu of S. aureus, and infection status was evaluated 24 h later following euthanasia. All infused glands of non-transgenic mice were infected, while glands form the high expressing transgenic mice were free of bacteria. Glands from the low and medium lines were substantially protected although some glands still contained bacteria from the substantial challenge dose. Thus, the transgenic mice have substantial resistance to S. aureus mastitis. We have performed preliminary evaluation the mammary development and lactational ability of the transgenic
mice. An unexpected finding is that the transgenic mice do not appear to have the same lactational ability as non-transgenic mice. Non-transgenic pups were cross-fostered onto either transgenic or novel non-transgenic dams. The growth of the pups was then followed. The growth rate of non-transgenic pups suckling transgenic dams was less than that of non-transgenic pups suckling non-transgenic dams. However, by three months of age the transgenic animals have normal body weights. Transgenic cows with the same lysostaphin-containing transgene have now been produced. Final data analysis on the their ability to secrete lysostaphin into milk, and mastitis resistance is being completed and will be submitted for publication in the near future. In the current transgenic animals, the lactation-specific B-lactoglobulin promoter region controls lysostaphin production. Therefore, the animals will produce lysostaphin during the lactation period. We now want to extend coverage of protection into the
mastitis susceptible non-lactating period through use of the lactoferrin gene promoter. This promoter is active in the non-lactating mouse mammary gland, and is induced by involution and infection. We have now cloned 4.4 kb of the bovine lactoferrin promoter region (Genbank Accession # AY319306) and constructed a transgene directing expression of lysostaphin. The transgene is functional when transfected into HC11 mouse mammary cells in vitro. In summary, experiments conducted during this project clearly demonstrated the application of transgenic technology to enhancing animal disease resistance. Transgenic cows containing the lactation specific transgene that was effective in transgenic mice have been produced. We have begun work on a new gene construct containing the bovine lactoferrin promoter.
IMPACT: 2000-12-01 TO 2004-11-30
Dairy cows with enhanced mastitis resistance would require less use of conventional antibiotics to treat this disease. A reduction in mastitis will also enhance the quality of milk being delivered to the processor, and will improve the welfare of the animals.
PUBLICATION INFORMATION: 2000-12-01 TO 2004-11-30
Powell, A.M., Talbot, N.C., Wells, K.D., Kerr, D.E., Pursel, V.G., and Wall, R.J. 2004. Cell donor influences success of producing cattle by somatic cell nuclear transfer. Biology of Reproduction 71:210-216.
PROJECT CONTACT INFORMATION
| NAME: |
Kerr, D. E. |
| PHONE: |
802-656-2113 |
| FAX: |
802-656-8196 |
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