Progress 11/01/04 to 09/30/07
Outputs
OUTPUTS: The goal of this project was to determine whether sperm-mediated DNA delivery is suitable for generation of transgenic chickens. For the final year of this project our specific aims were to: 1) continue screening of progeny from transfected sperm, 2) develop two reporter constructs for expression in transgenic chickens, and 3) investigate adoption of cultured primordial germ cells (PGC). Aim 1 was completed in spring 2007 with the genotyping of over 200 chicks from a large scale sperm transfection. We identified 15 chicks with potential weak positive PCR product for the transgene (LacZ) from the initial blood samples. These chicks were then sampled for blood, and comb, for re-screening. No positives were found in the re-screening. Therefore, we conclude that, despite our best efforts, that sperm-mediated gene transfer is either highly inefficient or incapable of generating stable transgenics. Aim 2 has continued after termination of the project. We have fused the CRE gene to
a constitutive promoter from pMIdeltaZ. The next step is to move this cassette into a retroviral packaging vector, pFB. Unfortunately, the undergraduate student for this project was awarded a study-abroad program in Australia for January through July. In September the effort was renewed. Our other construct was to clone a 6 kbp region from the chicken DAZl gene and fuse this to a GFP promoter. Expression of the DAZl promoter should be restricted to PGCs and perhaps other germ cells. We obtained BAC clones spanning the DAZl region, and designed PCR primers for obtaining overlapping products. To date we have not obtained the entire 6 kbp fragment as one product, but have trimmed the region from 174 kbp down to 12 kbp. That work continues and the GFP cassette is already in pFB. Aim 3 utilized a collaborator of the PI, Dr. J. Petitte at NCSU. That group has duplicated the culturing of PGCs. Dr. Petitte has offered to share the protocols and techniques and host Dr. Rhoads in mastering the
culturing protocols. According to published protocols, cultured PGCs can be transfected in culture, and then re-introduced into recipient chick embryos, to generate germline transformants (Lavoir et al., 2006). Dr. Petitte has volunteered to introduce our constructs into PGCs and assist us in the transgenesis. Once we have established PGC methods, Dr. Petitte has offered to help us start our own PGC cultures (predicated on additional funding). Our goal remains to demonstrate introduction of these 2 genes into chickens, expression of both transgenes in a wide range of tissues, and inheritance of this same phenotype in subsequent generations (i.e., germline transmission). Once we have developed reliable transgenesis we will need to design additional vectors with different tissue specific promoters to direct expression to specific tissues (e.g., oviduct, hypothalmus, epithelium, testis). These promoters can be obtained by PCR using the chicken genome asembly and then used to replace the
promoter region in pMIEM or pMIΔZ. Transgenic chicks will be examined for correct tissue expression.
PARTICIPANTS: University of Arkansas: Dr. Walter Bottje, Dr. Douglas Rhoads, Ms. Brenda Flack, Mr. Phillip Cleves, Ms. Candace Smith. Collaborators: Dr. David Froman- Oregon State University; Dr. James Petitte- North Carolina State University.
Impacts
The ability to generate transgenic chickens expressing different genes and not limited to viral based delivery or limited in expression to particular tissues, will allow researchers to examine the roles of different genes in different disease processes. For example, Drs. Rhoads has a long history (previously funded by industry and USDA) on gene defects affecting male fertility. This research would be greatly enhanced through development of transgenic chickens. The ability to add genes or mutated genes to the germ line would allow us to manipulate the genome for assessment of the role of the gene in development and spermatogenesis. We completed an Arkansas Biosciences Institute funded EST project characterizing the transcriptome of the chicken reproductive tract. We have reported that we have detected over 400 unique genes expressed in the chicken reproductive tract (Froman et al., 2006). The PI has submitted a USDA-NRICGP proposal and an NIH R15 proposal to followup on
this project for detailed characterization of the sequences of these unique genes and the specificity of tissue expression. We anticipate that some of these genes are regulatory RNAs in the newly recognized class of long non-coding RNAs (Goodrich and Kugel, 2006). Availability of means to introduce transgenes will allow us to generate transgenic chickens with altered potential long non-coding RNAs to determine the effects on development. We have been collaborating with Dr. Jim Petitte at NCSU on developing vectors and genes to address the developmental role of specific genes in contribution to the germ cells of the gonad (PGCs). Transgenesis is essential to manipulating the chicken genome to determine the significance of developmental time points, and particular cell populations to the germ line. Through our SDD research (supported by the poultry industry) and the basic research questions on male gonadal development, we are moving into areas of interest to NSF and NIH-NIDDK. Our goal
is to establish reliable means to use the chicken as a medical model for developmental aspects of gonadal development. Similar approaches for transgenesis could be applied to poultry transmitted bacterial food pathogens in collaboration with UA and ARS researchers. Others have reported that cytokines affect carriage of salmonella (Swaggerty et al., 2004). Cytokine genes under control of alternative promoters could be introduced to examine the role of cytokine induction in bacterial infection. Similar approaches could be used to examine susceptibility to viruses, or physiological diseases like ascites or skeletal abnormalities. As scientists identify or develop potential genes for these diseases, they can be introduced into chickens to examine affects on the disease or health parameter.
Publications
- Froman, D. P., Kirby, J. D., and Rhoads, D. D. (2006). An expressed sequence tag analysis of the chicken reproductive tract transcriptome. Poultry Science 85, 1438-1441.
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Progress 01/01/06 to 12/31/06
Outputs
The goal of this project is to determine whether sperm-mediated DNA delivery is suitable for generation of transgenic chickens. We have determined that DNA can be introduced into the sperm using liposome formulations. We optimized the procedures to maximize DNA uptake while maintaining sperm viability (mobility). We then moved to artificial insemination using liposome transfected semen. Initial experiments showed significant declines in percentage of fertile eggs (from 90% decline to 30%). We therefore investigated the amount of DNA and transfection time, optimizing for amount of DNA and embryo viability. We were able to vastly increase the number of fertile eggs that developed to hatch. We generated 196 offspring that were generated with sperm transfected with the plasmid pMIEM which expresses Green Fluorescent Protein (GFP) under control of strong viral and chicken (actin, delta-crystallin) promoters. This construct has been shown by others to express in cell
cultures from a variety of tissues. We are presently screening these offspring using PCR tests we developed for GFP. Any chicks showing presence of the transgene will be further analyzed for GFP and LacZ expression in skin and wattle biopsies. Any chicks showing transgene expression will be raised to puberty and then used for production of offspring. Once offspring have been generated and screened (using the same procedures as the first generation), we will sacrifice the parent transgenics and assay a variety of internal tissues for expression. Our goal is to demonstrate introduction of genes into chickens, expression of both transgenes in a wide range of tissues, and inheritance of this same phenotype in subsequent generations (i.e., germline transmission). As a backup plan we intend to learn how to culture primordial germ cells (PGCs) from chick embryos. Dr. James Petitte at North Carolina State University has been successful in this regard and has agreed to consult. We have also
begun to construct a defective retroviral vector with the Ds-Red reporter gene under control of the DAZL gene promoter. DAZL is a PGC specific promoter so this vector should express DsRed only in PGCs. This should allow us to transfect embryos and the isolate cultured cells and sort those for PGCs. Cultured PGCs have been shown by others to be suitable for DNA transfection and then injection back into embryos to generate germ-line transgenic chickens.
Impacts
Other than the basic science aspects of the proposed work, useful real world applications could also be realized. If we are able to generate transgenic chickens then we can collaborate with other UA and ARS researchers on development of transgenic chickens with altered susceptibility to bacterial and viral pathogens. Others have reported that cytokines affect carriage of salmonella. Cytokine genes under control of alternative promoters could be introduced to examine the role of cytokine induction in bacterial infection. Similar approaches could be used to examine susceptibility to viruses, or physiological diseases like ascites or skeletal abnormalities. As scientists identify or develop potential genes for these diseases, they can be introduced into chickens to examine affects on the disease or health parameter.
Publications
- No publications reported this period
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Progress 01/01/05 to 12/31/05
Outputs
We propose to evaluate and extend this method of sperm-based delivery of transgenes into the chicken germ line. Two plasmids will be introduced, pMIEM expresses Green Fluorescent Protein (GFP) and pMIWZ expresses beta-galactosidase. Both genes are under control of strong viral promoters and both reporter genes contain additional chicken promoters (actin, delta-crystallin) for expression in cell cultures from a variety of tissues. The DNA will be introduced into sperm using liposomes. Once we have optimized the liposome mixture and sperm treatment the DNA treated sperm will be used to inseminate hens. Offspring will be screened using PCR tests for GFP and LacZ on DNA isolated from blood. Any chicks showing presence of the transgene will be further analyzed for GFP and LacZ expression in skin and wattle biopsies. Any chicks showing transgene expression will be raised to puberty and then used for production of offspring. Once offspring have been generated and screened
(using the same procedures as the first generation), we will sacrifice the parent transgenics and assay a variety of internal tissues for expression. Our goal is to demonstrate introduction of these 2 genes into chickens, expression of both transgenes in a wide range of tissues, and inheritance of this same phenotype in subsequent generations (i.e., germline transmission). Once we have developed reliable transgenesis we will need to design additional vectors with different tissue specific promoters to direct expression to specific tissues (e.g., oviduct, hypothalmus, epithelium, testis). These promoters can be obtained by PCR and then used to replace the promoter region in pMIEM and/or pMIWZ. Transgenic chicks will be examined for correct tissue expression. The ability to generate transgenic chickens expressing different genes and not limited to viral based delivery or limited in expression to particular tissues, will allow researchers to examine the roles of different genes in
different disease processes. This would be extended to our work on gene defects affecting male fertility. This research would be greatly enhanced through development of transgenic chickens. We would like to adapt organ co-culture using techniques demonstrated in mice. Co-culture of mesenchyme and mesonephros tissue with the embryonic gonad has been used to demonstrate migration of cells into the testis and formation of the epididymis. If we can generate analogous chickens expressing beta-gal we can adapt organ co-culture for analysis of epididymal developmental defects in SDD chickens. Normal gonad would be co-cultured with SDD mesenchyme and mesonephros. Conversely, SDD gonad would be co-cultured with normal mesenchyme and mesonephros. Epididymis formation and mesenchymal cell migration into the testis can then be monitored for affects of the SDD mutation. Thus we could determine whether the SDD defect depends on signals from the gonad or the mesenchyme and which tissues contribute
to the signals. Organ co-culture would allow us to move our SDD research from a fertility problem affecting the poultry industry into basic research questions on male gonadal development.
Impacts
Other than the basic science aspects of the proposed work, useful real world applications could also be done. Similar approaches could be applied to poultry transmitted bacterial food pathogens in collaboration with UA and ARS researchers. Others have reported that cytokines affect carriage of salmonella. Cytokine genes under control of alternative promoters could be introduced to examine the role of cytokine induction in bacterial infection. Similar approaches could be used to examine susceptibility to viruses, or physiological diseases like ascites or skeletal abnormalities. As scientists identify or develop potential genes for these diseases, they can be introduced into chickens to examine affects on the disease or health parameter
Publications
- No publications reported this period
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