GOATS PRODUCING SPIDER SILK PROTEINS IN THEIR MILK

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0213768
• Project Start Date: Jan 1, 2008
• Project End Date: Dec 31, 2010
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF WYOMING
1000 E UNIVERSITY AVE DEPARTMENT 3434
LARAMIE,WY 82071-2000

Performing Department
MOLECULAR BIOLOGY

Non Technical Summary
High level production of any recombinant protein is a major challenge. The use of either animals or plants for production would substantially benefit the agricultural community. We are currently implementing the production of spider silk proteins in alfalfa. However, this is clearly a long-term project and in view of the recent court decision preventing the planting of Round-up Ready Alfalfa there maybe serious impediments to using that system for large-scale production. In collaboration with Nexia Biotechnologies of Montreal, Canada we have generated transgenic goats that produce the spider silk proteins in their milk. These goats are expressing spider silk proteins in the milk.

Animal Health Component

40%

Research Effort Categories

Basic

50%

Applied

40%

Developmental

10%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
301	3820	1040	75%
303	3820	1080	25%

Knowledge Area
303 - Genetic Improvement of Animals; 301 - Reproductive Performance of Animals;

Subject Of Investigation
3820 - Goats, meat, and mohair;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

spider silk

milk

transgenic goats

protein

fiber spinning

Goals / Objectives
The objective is to have a herd of transgenic goats producing spider silk proteins in their milk at UW in the next 18 months. This will be achieved in one of three ways. The first and easiest is if we can import the goats from Canada. If that is fails then the second approach will be to inseminate goats with sperm from the founder males of the Nexia herd. Finally, if all else fails we will collaborate with University of California, Davis to clone the goats.

Project Methods
There are three possible routes. We already have a preliminary INAD permit from the FDA who has jurisdiction over transgenic animals. The details of the restriction on the permit will evolve as we progress with the project but I have discussed all of the approaches with them in a meeting in May. The first is importation of five goats for each of the two key spider silk proteins that have shown the ability to produce them in the milk in useful quantities. In order to group the kidding dates we will use CIDR implants for ovulation. The only difficulty is that the USDA currently bans importation of all ruminants for non-slaughter purposes from Canada. I have requested a research exemption to the ban from Dr. John Clifford, Assistant Deputy Administrator, Veterinary Services, APHIS, USDA. The second approach will be to purchase goats in the US and breed them using semen from the two founder males that generated the current Nexia herd. We have already identified sources to obtain the needed 20 goats for the project. The semen has been frozen for over three years thus we will use laproscopic artificial insemination as opposed to transvaginal as the motility is very likely compromised by the freezing and further by long term storage. The semen will be provided by Nexia. This will be done in collaboration with Dr. Bill Murdoch, who has agreed to conduct the procedure for us. We plan to wean the kids at 60 days and then rebreed the does as soon as possible after that to increase the number of female offspring. The keys that are less worked out are the insemination timing as there are only a few papers that have used synchronization rather than teaser bucks. The timing is reasonably consistent but using the CIDRs may change that slightly but we will insure that we inseminate prior to ovulation to insure placement of the semen. We will use ultrasound to determine pregnancy status and rebreed any goats found to be open as the heat cycle will be established by the CIDR implants. This third approach, which could be conducted independently, but more likely would be in conjunction with the breeding approach is to clone the current females using tissue samples from them. This work will be done by Dr. Jim Murray, U. of California, Davis, who has agreed to conduct this work if required. He has successfully cloned goats as well as generated transgenic goats expressing other proteins in the milk. There are two concerns with this approach. The first is getting the tissue from Canada in a sufficiently pristine condition to allow cloning. The most likely scenario would be to collect the tissue and fly immediately to UC Davis with the tissue in culture media kept cold. The possible problem is related to the one noted for breeding and that is the integrity of gene passage as that has not been studied from these females either. That seems less of a problem here as the gene stability is usually excellent in the direct cloning from differentiated tissue.

Progress 01/01/08 to 12/31/10

Outputs
OUTPUTS: 1. Substantial quantities of milk containing both spider silk proteins was collected. 2. Substantial amounts of both spider silk proteins were purified. 3. New purification protocols were developed. PARTICIPANTS: 1)Randolph V. Lewis, PI 2) Justin Jones, Research Scientist, overall responsibility for goat husbandry 3) Holly Steinkraus, Research Scientist, helped with goat care 4) Heather Rothfuss, Research Scientist, responsible for purification of proteins. TARGET AUDIENCES: Other researchers. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
1. The new purification protocols led to higher yields and purity than previously published protocols. 2. There was a large variation in both milk and protein production among the goats, surprisingly even between clones.

Publications

• No publications reported this period

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: The goats were bred, kidded and milked for two rounds. Milk was used to purify the two major spider silk proteins. Non-transgenic does bred to transgenic bucks were tested before, during and after gestation for the presence of the transgene in the blood. Protein is being spun into fibers and has been given to a collaborator for testing for reinforcement of a bone replacement matrix. We found no evidence for the transgene using PCR in any of the goats at any time. We showed that our detection protocol was sensitive enough to detect the transgene in at a dilution of 1:50,000 into normal goat blood. PARTICIPANTS: R.V. Lewis: Professor; Justin Jones: Research Scientist; Holly Steinkraus: Research Scientist; Mathew Assay: Undergraduate Pre-veterinary student TARGET AUDIENCES: NSF, FDA, NIH, DOD, and various medical device manufacturers. PROJECT MODIFICATIONS: We were able to negotiate the "purchase" of the rest of the goats held in Canada due to the closure of the company that developed them.

Impacts
We have had two film crews out to film the goats, the milk collection and the proteins purification and fiber formation from NSF and from NOVA. We will be sharing the results of the blood tests with the FDA who regulates the transgenic goats.

Publications

• No publications reported this period

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Protein biomarkers of CWD were identified through the purchase and implementation of a new, state-of-the-art proteomics instrument, the Beckman PF2D. The use of the PF2D to identify potential biomarkers of (CWD) that could potentially be used as ante-mortem indicators of the diseases provides more-comprehensive information than does traditional 2D gel electrophoresis. The PF2D is a liquid means by which to separate proteomes into useful and manageable fractions that are readily submitted for trypsin digests and mass-spectrometry analysis for peptide mapping and identification. A proteome is initially separated via cation exchange, and is then further separated using reversed-phase chromatography-thus providing 2-D separations. Beginning with 2 mg of concentrated deer urine protein per proteome fractionation, the instrument keeps the proteins in solution throughout the process and allows for very selective and precise quantity differences to be observed. We successfully performed proteome fractionations on both negative and positive deer urine. We began by pooling urine from 5 negative animals and separately pooling urine from 5 positive animals. The pooled negative urine and the pooled positive were separately fractionated and the results compared. Proteins significantly up-regulated in the positive animals were the only proteins selected for further study. One of the primary considerations when looking for protein biomarkers is that their blood concentration are significantly different from pre-infection levels to reduce error in determinations and aid in the ease of detection. Proteins determined to meet these criteria were trypsin digested and subjected to mass fingerprint analysis for identification. Considering that the deer genome is unknown, the identification and analysis had to be performed based upon likely homologies to other known mammalian proteins. This method worked very well, and we have successfully identified 9 proteins that are significantly over-expressed in the diseased state. To date, we have accomplished an extensive analysis of urine from CWD-positive animals. The analysis has identified 9 potential biomarkers, as represented in Table 1. Urine is not an ideal source of biomarkers, nor is it an ideal fluid to test for the presence of the disease-but we feel strongly that markers found in the urine will also be present in the serum and other fluids of infected animals and our preliminary results are bearing this out. Additionally, when compared to serum, there is a low abundance of confounding proteins such as serum albumin and immunoglobulin's that mask the presence of other proteins. The removal of those proteins from deer serum proved difficult, and somewhat unreliable, and we decided to move in another direction while working on the serum clearance procedures. PARTICIPANTS: Randolph V. Lewis, professor; Justin Jones, research scientist; Benjamin Brooks, graduate student, Ted John, research scientist; Wyoming Game & Fish Department; Colorado Fish and Wildlife; USDA; Washington State University. TARGET AUDIENCES: Other scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The outcome of our work to identify a specific response to TSE has been the identification of 9 potential biomarkers to CWD. The real impact is, that if these biomarkers work the way we expect then monitoring CWD will become significantly easier. A test using multiple biomarkers to eliminate false positive and negatives and that is performed in the field is envisioned. This compared to our current methodologies of removing lymph tissues, brain matter, or cerebral spinal fluid is very beneficial. Additionally, the test formats for protein biomarkers are simple and able to be accomplished by non-technical personnel thus not limiting testing to very skilled laboratory personnel. Work is currently underway to observe the biomarkers in fecal matter. A fecal matter test for CWD would be ideal to identify the scope of CWD and to help monitor its spread. Preliminary work is very promising. Additionally, it is expected that these biomarkers will not be species specific and that we will be able to observe them in other TSE's. This would have a tremendous impact on our ability to test animals for Bovine Spongiform Encephalopathy as well as to test for the human form(s) vCJD and CJD.

Publications

• Brooks, Benjamin D.,Albertson, Amy E., Jones, Justin A., Speare, Jonathan O., Lewis, Randolph V. 2008. Efficient screening of high-signal and low-background antibody pairs in the bio-bar code assay using prion protein as the target, Analytical Biochemistry 382: 60-62