Production of hA20-transgenic pigs by somatic cell nuclear transfer for xenotransplantation research

Medientyp: E-Book; Hochschulschrift

Titel: Production of hA20-transgenic pigs by somatic cell nuclear transfer for xenotransplantation research

Beteiligte: Oropeza, Marianne [Sonstige Person, Familie und Körperschaft]

Erschienen: 2009

Umfang: Online-Ressource (VII, 148 S. = 47.150 Kb, text)

Sprache: Englisch

Identifikator:

Schlagwörter: Schwein > Transgene Tiere > Heterotransplantation > Expressionsvektor > Klonierung

Entstehung:

Hochschulschrift: Zugl.: Hannover, Tierärztl. Hochsch., Diss., 2009

Anmerkungen:

Beschreibung: Schwein, Xenotransplantation, Transgen. - The goal of this project was to produce hA20-transgenic pigs by somatic nuclear transfer (SCNT) with protective features for xenotransplantation. The first experimental phase included design and construction of two hA20 expression constructs (pCAGGSEhA20-IRESNEO and pEF1/HisAhA20). In the second phase porcine fetal fibroblasts (pffs) were isolated and transfected with the expression vectors. This was followed by SCNT cloning with hA20-transgenic pffs as donor cells. In the final phase of the project transgenic fetuses and piglets were analysed for hA20 expression and functionality of the transgene. The following results were obtained: 1) The production of two hA20 expression vectors was successful, and large amounts of each vector were produced for transfection experiments. 2) Transfection of pffs with pCAGGSEhA20-IRESNEO resulted in 82 resistant cell colonies. Five cell colonies were selected for SCNT experiments. A total of 12 sows served as recipient animals and 9 pregnancies were established (75%). The transfer of a total of 1036 cloned embryos yielded 52 fetuses and piglets, a cloning efficiency of 5%. Twelve of 52 fetuses and piglets (23.1%) were transgenic. Recloning with pffs derived from pCAGGSEhA20-IRESNEO-transgenic fetuses resulted in 3 out of 4 pregnant recipients (75%). Recloning with pffs from transgenic fetuses resulted in a cloning efficiency of 2.3%. Nine cloned transgenic piglets were born. 3) Transfection of pffs with the pEF1/HisAhA20 construct resulted in 33 resistant cell colonies. Nine of the colonies were selected for SCNT. Embryos were transferred to 4 recipients and three pregnancies were established (75%). A total of 375 cloned embryos resulted in fifteen fetuses and piglets, representing a cloning efficiency of 4%. Twelve out of the fifteen progeny had integrated the expression vector (80%). Recloning sessions with pffs derived from pEF1/HisAhA20-transgenic fetuses resulted in pregnancies in two out of three recipients (66.6%). Cloning efficiency after recloning was 1.5% (four born piglets). 4) Transgenic animals were analysed for mRNA and protein expression of hA20 by reverse transcriptase polymerase chain reaction (RT-PCR), Real-time PCR, Northern blotting, immunohistochemical staining and Western blotting. HA20 was expressed in skeletal muscle, heart, and importantly in freshly isolated and cultivated porcine aortic endothelial cells (PAECs) in pCAGGSEhA20-IRESNEO transgenic pigs. 5) Functional in vitro assays using cultivated PAECs, revealed that the hA20-transgenic PAECS were protected against apoptotis after challenge with human tumor necrosis factor alpha (hTNF-α) compared with wildtype cells. No significant differences were observed in the expression of selected endothelial activation markers, such as intercellular adhesion molecule-I (ICAM-I), vascular adhesion molecule-I (VCAM-I) and E-selectin after exposure to hTNF-α. 6) Four hA20 porcine hearts were evaluated in an ischemia-reperfusion injury model and compared with three wildtype controls. Subendocardial segment shortening (SES) was significantly different between the two groups. The hA20-transgenic animals showed better cardiac performance than wildtype animals. This is the first study reporting the production of hA20-transgenic pigs, which represent an important step in the production of multi-transgenic pigs for use in xenotransplantation experiments. The hA20 transgenic piglets developed normally. Improvements may be obtained with improved expression vectors to target hA20 expression to the endothelial cell layer. Encouraging results were obtained with respect to cloning efficiency, which ranged between 1.5 and 5% depending on the donor cells. These results were above the normal cloning efficiency in pigs which does not exceed ~1%. Cells and tissue from pigs expressing hA20 were subjected to in vitro and in vivo functional tests and results partially confirmed previously published results. Overall the results of this thesis demonstrate that A20 is an important candidate gene for future xenotransplantation research. The next step will involve production of multi-transgenic pigs on the basis of an α 1,3-galactosyltransferase gene knockout. These pigs should control both hyperacute rejection (HAR) and acute vascular rejection (AVR) by transgenic expression of hA20.

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