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Open access

Joanna Zeyland, Daniel Lipiński and Ryszard Słomski

Abstract

Mechanisms regulating the activity of the complement system responsible for the rejection of transplant organs are balanced so that the attack is instantaneous but is restricted to the infected cells of the organism. The most important components regulating its activity comprise CD55 and CD46 factors as well as the CD59 anchored in the cell membrane. Hyperacute response of the immunological system appears to be the key in the xenotransplant rejection and the elaboration of methods preventing its occurrence will give a real chance for the development of xenotransplantation.

Gene constructs containing coding sequences of human CD46, CD55 and CD59 were prepared and used to transfect porcine fetal fibroblasts. Stable lines were molecularly characterized for an integration of transgenes by PCR. Lines with a stable integration of transgenes were subjected to further characterization of expression by RT-PCR and vitality test. Molecular characteristics of the transgenic cell lines obtained revealed a steadfast integration and, in the majority of cases, expression of the introduced transgenes. The performed cytotoxicity analysis demonstrated that transgenic lines were characterised by a higher survivability rate than non-transgenic cells in the presence of human serum which proved their protective influence in relation to the activity of the complement system.

Open access

Daniel Lipiński, Joanna Zeyland, Andrzej Pławski and Ryszard Słomski

Determination of the Absolute Number of Transgene Copies in CMVFUT Transgenic Pigs

The aim of this research was to determine the number of transgene copies in the DNA of transgenic pigs. The copy number of the transgene was analysed in the transgenic animals with introduced pCMVFUT genetic construct containing a coding sequence of human H transferase under a control of CMV promoter. The copy number of the transgene that had integrated with the genome of the transgenic animals was analysed by qPCR with SYBR Green dye, which enabled nonspecific double-stranded DNA detection. CMVFT-2F and CMVFT-2R primers were used to amplify a 149 bp fragment of DNA. Forward primer had a sequence complementary to a promoter sequence and reverse primer to a coding sequence of H transferase. The copy number of the transgene in the examined samples was established by plotting the CT values obtained on a standard curve, which had been set by the usage of the CT values for the successive standard dilutions with known copy number (1.438-1.431 copies). As a standard we used pCMVFut genetic construct hydrolyzed with Not I restriction enzyme to a linear form. The real-time PCR results helped to establish the range of 3 - 4 as the number of the transgene copies that had integrated to the swine genome.

Open access

Jacek Jura, Zdzisław Smorąg, Barbara Gajda, Daniel Lipiński and Ryszard Słomski

Abstract

Possible influence of a transgene on life functions of embryos makes it reasonable to confirm or deny it for a particular gene construct. In vitro development of an embryo is a widely used criterion of its competence. The aim of the study was to compare in vitro developmental capacity of transgenic and non-transgenic pig embryos. The results showed a statistically significant difference in in

vitro developmental capacity of embryos obtained from transgenic and non-transgenic pigs. Developmental competence of embryos (morula and blastocyst stage) produced from zygotes obtained from transgenic sows decreased compared to that obtained from non-transgenic sows.

Open access

Jolanta Opiela, Daniel Lipiński, Joanna Romanek, Wojciech Juzwa, Michał Bochenek and Piotr Wilczek

Abstract

Mesenchymal stem cell (MSC) differentiation is regulated intrinsically by transcription factors and extrinsically by the extracellular matrix. We tested whether matrix metalloproteinase-2 (MMP-2) and its inhibitor TIMP-2, MEF2a and TAZ transcription factors are involved in porcine MSC differentiation towards adipocytes and osteocytes. Flow cytometry and immunofluorescence were used to investigate the expression levels of multipotent cell surface markers CD73 and CD105. Real- time PCR was performed to detect the osteogenic- and adipogenic-specific markers, osteocalcin and aP2, respectively, and to estimate the MMP-2, TIMP-2, MEF2a and TAZ transcript expression levels in three groups of cell, i.e., undifferentiated MSCs, adipocytes (A) and osteocytes (O). We showed that at the transcript level, the differentiation of MSCs towards adipocyte fate may involve MMP-2, TIMP-2 and TAZ. We also show that the differentiation of MSCs toward osteocyte fate may involve TIMP-2, MEF2a and TAZ. Our research provides preliminary data on the possible role of the MMP-2, TIMP-2 and TAZ transcripts in adipogenic differentiation and of the TIMP-2, TAZ and MEF2a transcripts in the osteogenic differentiation of porcine MSCs. We report for the first time the possible involvement of MEF2a in the osteogenesis of porcine MSCs. Our work may provide additional evidence for the MMP-independent function of TIMP-2 during osteogenesis.

Open access

Magdalena Hryhorowicz, Joanna Zeyland, Agnieszka Nowak-Terpiłowska, Jacek Jura, Wojciech Juzwa, Ryszard Słomski, Jan Bocianowski, Zdzisław Smorąg, Anna Woźniak and Daniel Lipiński

Abstract

The use of pigs as a source of organs and tissues for xenotransplantation can overcome the growing shortage of human donors. Human NK cells play an important role in the cell-mediated rejection of pig-to-human xenografts. In this paper we report the generation and extensive characterization of three generations of transgenic pigs with HLA-E gene encoding the antigen which can inhibit the human NK cell-mediated response. The gene construct pHLAE-GFPBsd containing the human gene encoding the human leukocyte antigen under the promoter of the EF-1α elongation factor ensuring systemic expression was introduced by microinjection into a pronucleus of the fertilized porcine oocyte. PCR analysis revealed and FISH analysis confirmed that the pHLAE-GFPBsd gene construct was present in the genome of the founder female pig. As a result of inter-breeding, an additional 7 transgenic animals were obtained (one individual from F1 generation and six individuals from F2 generation). The transgene expression was shown by RT-PCR and flow cytometry. Real Time PCR analysis estimated the approximate number of transgene copies at 16–34. Karyotype analysis did not show any changes in the structure or the number of chromosomes. The expression level of the transgene was stable in the next generation of genetically modified pigs. An NK cell-mediated cytotoxicity assay showed the increased viability of the transgenic cells in comparison with the wild-type, which confirmed the protective influence of HLA-E expression.