Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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16 t h International Congress on Animal Reproduction 204 Poster Abstracts Conclusions These preliminary data suggest that flow cytometry could be used for evaluating sperm DNA binding capacity. Besides this study shows that under our experimental conditions the capacity of binding of DNA to bull frozen-thawed bull spermatozoa is related to viable and unviable cells. These facts maintain opened the possibility of using this technique in IVF systems for generation of transgenic bovine embryos. Supported by BIOCARM 10BIO2005/01-6463 and MEC-FEDER AGL2006-03495 P534 On the role of LIF and LIFR expression during rabbit embryonic stem cell line establishment Gócza, E.*, Catunda, AP., Hiripi, L., Bősze, Zs. Agricultural Biotechnology Center, 2100, Szent-Györgyi A. str. 4, Gödöllő, Hungary Embryonic stem cells (ES) have become an important tool for generating transgenic mice. Embryonic like stem cells and ES chimeric animals have been reported for several farm animal species, but germ line transmission was not reported. Rabbit ES cells, established in our laboratory, by their growth in tightly packed, flat colonies resembled to human ES cell lines, while expression of alkaline phosphatase, Oct-4, Nanog, SSEA-1 and CD9 which were detected by immunostaining made them similar to mouse ES cells. Leukemia inhibitory factor (LIF) is necessary for mouse ES cell selfrenewal, but it fails to maintain self-renewal in human and non-human primate ES cells. To examine the role of LIF and leukemia inhibitory factor receptor (LIFR) at rabbit ES cell line establishment, the expression patterns of LIF and LIFR were analysed at different stages of rabbit embryonic development and LIFR expression in rabbit ES cells by RT-PCR at different passages. LIFR mRNA was first detected around implantation in 6.5 d.p.c. rabbit embryos, while LIF mRNA expression was already present at rabbit blastocyst stage. In attached ICM and in ES cells after the 1st passage, very low level of LIFR expression was found. After the 2nd passage high expression of LIFR was detected in rabbit ES cells. Since commercial rabbit LIF is not available we had to find the most effective recombinant LIF for rabbit ES cell establishment. With this object human, rat and mouse recombinant LIFs were compared. At early passages we could not find any difference in their effect, but at higher passage numbers, the rat LIF was found to be the most efficient factor in the prevention of the differentiation. LIF withdrawal resulted the differentiation of rabbit ES like cells into cardiomyocytes. These findings suggest that the the LIF-LIFR signal pathway has an important role in maintenance of pluripotency of rabbit ES cells. This work was supported by grant OM-00367/2004, OTKA T037582 and Bilateral Intergovernmental S and T Cooperation D-26/02 (HUN02/045). P535 Succesful h-lactoferrin transgenic goat embryo transfer after SCNT Mutto, A 1 * # ; Kaiser, GG 2# ; Mucci, N 2# ; Hozbor, F 2 ; Sanchez, E 2 ; Ugalde, R 1 ; Alberio, RH 2 1IIB, UNSaM, Argentina; 2 INTA Balcarce, Argentina #Authors contributed equally to the present work Because of its properties (digestibility, composition, taste), goat milk is suitable to be used as a substitute for cow milk in patients with cow milk allergy and for human nursling babies with no tolerance to lactose. By adding some sugars, lisozime C and lactoferrin it will be possible to have a “humanized” goat milk. The objective of this work was to produce transgenic clone embryos and pregnancies for human lactoferrin in goats by using somatic cell nuclear transfer of transfected fetal goat fibroblasts. Materials and methods A 1960bp human lactoferrin cDNA (Gen Bank n: NM_002343) obtained from ATCC encompassing the entire coding region was used as template. It was cloned into pBC1 milk expression vector (Invitrogen, Ca, USA) between the intron 1, exon 2 and exon 7 of goat β-casein gene. An eukaryotic selection marker cassette with a Blasticidin-S resistence gene was added, under h-EF1α promotor. Goat fetal fibroblasts obtained from 30 day pregnancies were sexed by PCR and karyotyped and female cell lines were transfected by liposomes (Lipofectamine 2000®, Invitrogen, USA). Colonies were selected after 10 days of culture with Blasticidin and grown in complete culture media (D-MEM). Stable transfections were confirmed by FISH. Oocytes were obtained by laparoscopic ovum pick-up of stimulated goats during the reproductive season, in vitro matured and subjected to nuclear transfer as described by Baldassarre et al (2004). Presumptive zygotes (24 hs after NT) were transfered by a surgical procedure in both oviducts of those animals that presented at least 2 hemorrhagic corpora lutei. Results A total number of 331 oocytes were recovered from 480 follicles after 5 sessions of LOPU (8 goats each session). All oocytes that presented a polar body were enucleated and transferred (n=238). There was an overall fusion rate of 51%. A total number of 121 presumptive zygotes were transfered to 12 goats (7 to 12 per recipient), resulting in 5 pregnancies at day 30 (41.6% pregnancy rate) and 2 at day 83 (16.6%). Two pregnancies were twins, one remaining until day 83. We can conclude that under our conditions it is posible to obtain transgenic goat fetal fibroblasts, embryos and pregnancies for human lactoferrin. For our best knowledge this is the first report of goat h- lactoferrin transgenic pregnancies by using SCNT technique. P536 Towards generation of human A20 gene transgenic pigs with improved features in xenotransplantation Oropeza, M 1,2 *, Petersen, B 1 , Herrmann, D 1 , Hassel, P 1 , Lucas-Hahn, A 1 , Lemme, E 1 , Queisser, AL 1 , Niemann, H 1 1Dept. of Biotechnology, Institute for Animal Breeding, Mariensee, Neustadt, Germany; 2 University of Veterinary Medicine, Hannover, Germany Introduction Xenotransplantation is considered as promising to close the growing gap between demand and availability of appropriate human organs. While the hyperacute rejection response can already be reliably controlled, the acute vascular rejection (AVR) remains a major hurdle for longterm survival of xenografts in a porcine-toprimate organ transplantation. The human A20 (hA20) gene exhibits both antiapoptotic and anti-inflammatory properties in endothelial cells (Transplantation 82 : 36-40, 2006) and could thus prevent endothelial cell activation leading to AVR and xenograft destruction. The goal of this project is to produce transgenic pigs with improved features in xenotransplantation by transgenic expression of hA20. Materials and Methods The hA20-expression vector driven by the ubiquitous CAGGS hybrid promoter (chicken β-actin-/ rabbit β- globin) containing an IRES-neomycin resistance cassette (9.1 kb) was transfected into porcine fetal fibroblasts (PFFs) derived from German Landrace porcine fetal explant cultures. Transfection of 3 x 10 6 cells was accomplished at 450 V and 350µF with 10 µg plasmid DNA. G418-resistant (800 µg/mL) cell clones were screened by PCR with hA20 specific primers for hA20-integration. Somatic cell nuclear transfer (SCNT) was performed as recently described (Cloning Stem Cells 7: 35-44, 2005). Results A total of 80 cell clones were hA20-positive in PCR screening from 4 rounds of transfection after a 14 days G418 selection period. 30 positive cell clones were re-selected for 14 days with G418. Four of these cell clones were used as donor cells for SCNT. Immediately after SCNT, reconstructed embryos were transferred to a total of 10 synchronized recipient sows. Ultrasound examination on day 23-25 of pregnancy confirmed established pregnancies in 7 of 10 recipient sows. Two animals were sacrified on day 30 of pregnancy and a total of 8 fetuses were isolated. PCR and Southern Blot analysis showed integration of the hA20 gene in 6 of 8 fetuses. Conclusions The hA20-vector can be integrated in PFFs and hA20 transgenic PFFs can successfully be used in SCNT to establish pregnancies. Further analysis will focus on the expression patterns in A20-positive cell clones and the biological function of hA20 in transgenic piglets. A second approach will be to introduce an
16 t h International Congress on Animal Reproduction Poster Abstracts 205 endothelial specific promoter active in pigs and thus target expression of hA20 to the endothelial cell layer. Providing the endothelial cells with a higher antiapoptotic potential could decrease their susceptibility to cell death. P537 Expression of an omega-3 fatty acid desaturase gene from scarlet flax in bovine transgenic embryos cloned from transfected somatic cells Saeki, K 1 *, Indo, Y 1 , Tatemizo, A 1 , Suzuki, I 2 , Matsumoto, K 1 , Hosoi, Y 1 , Murata, N 3 1Department of Genetic Engineering, School of Biology-Oriented Science and Technology, Kinki University, Japan; 2 Laboratory of Plant Physiology and Metabolism, University of Tsukuba, Japan; 3 National Institute for Basic Biology, Japan Introduction Long chain n-3 fatty acids are considered desirable in human diets because they can lower the risk of coronary artery disease, cancer and inflammatory diseases. n-3 fatty acids are found in fish oils and specific plant oils. But their levels in animal meats are quite low, because mammals lack the gene for converting linoleic acid to alfa-linolenic acid. Recently, it has been reported that a humanized nematode gene, hfat-1, can be used to increase the levels of n-3 fatty acids in transgenic pigs. We report here functional expression of a plant-derived gene for an omega-3 fatty acid desaturase (FAD3) in gene-transfected bovine cells and production of embryos cloned from the transfected cells. Methods The gene was isolated from immature seeds of scarlet flax, because the level of alfa-linolenic acid of the flax seeds is the highest among terrestorial plants. Bovine muscle satellite cells were isolated from a 9-month-old male calf. The codon usage of the flax FAD3 cDNA was optimized (humanized) for high expression in mammalian cells. A plasmid (pIRES2-EGFP) containing the humanized FAD3 gene (hFAD3) under the control of the CAG promoter, and a neomycin-resistance cassette (pCAG/hFAD3/IRES/EGFP/(neor)) was transfected to the satellite cells with a transfection reagent ( GeneJammer ). The stably transfected cells were differentiated to multilocular adipocytes by culturing with bFGF, dexamethasone and octanoic acid. Total lipids were isolated from the adipocytes and their fatty acid composition was analyzed by gas chromatography. We then produced cloned bovine embryos using the hFAD3 cells. The cloned embryos were cultured to the blastocyst stage. Blastocyst rates and gene expression of EGFP and hFAD3 were then examined. Results The level of total n-3 fatty acids in the hFAD3 cells (12.5%) was higher than that in the control cells (9.1%, P4 to 8 mm in diameter) sizes of ovarian follicles were transfected with liposome alone, both liposome and EGFP, or liposome and EGFP in combination with pEGISI respectively. The transfection procedure was performed according to the Lipofectamine2000 manufacturers’ instructions. Cell proliferation was quantified by the CellTiter 96® AQueous One Solution Cell Proliferation Assay. Cell apoptosis was detected using an Annexin V- FITC/Propidium Iodide for flow cytometry. Steroidogenesis was evaluated by measurements of both estradiol and progesterone in culture via radioimmunoassay methods. Maturation of oocytes cocultured with transfected GCs and subsequent embryo developments were also investigated. Results The transfection with pEGISI inhibited proliferation of GCs from medium follicles (P
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Reproduction in Domestic Animals - Facultad de Ciencias Veterinarias

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