Background: Somatic cell nuclear transfer is the method of choice to generate transgenic large animals. Success largely depends on a high expression level of the target protein. Selection of cell clones with the desired expression level is of paramount importance before nuclear transfer. In this work we compare different methods that can be used on a small number of cells and their predictive value. Methods: Two expression vectors for CD55 and CD39 were separately transfected to PK15 cell line. Following selection, five of the best growing clones resulting from each transfection were expanded and subjected to RT-PCR and Immunohistochemistry (IHC) analyses. For IHC analyses the mAB IA10 (BD-Pharmingen) – for CD55 – and the mAB BU61 (Ancell) – for CD39 – were used. The same antibodies were also used in Western blot (WB) analyses performed on samples subjected to non-reducing SDS–PAGE and electroblotted on PVDF membrane. The presence of the target transcripts was confirmed by Northern blot (NB) analyses using DIGlabeled probes (Roche). The proteins expression was also analysed by FACS conducted on chosen clones. Fibroblasts and PAECs deriving from one CD55–CD39 stillborn cloned piglet were subjected to IHC, NB and FACS analyses. Results: Three out of five (#24, #2 and #15) PK15–CD55 clones were positive to RT-PCR but only clone #24 was positive to IHC. Clone #24 was further analysed by NB, WB and FACS that confirmed the high expression level. Clone #2 revealed a low expression level by FACS not detected by IHC. All five PK15–CD39 clones were positive to IHC and RT-PCR analysis. Clone #10 was further analysed and confirmed positive by NB and WB. The IHC, NB and FACS data obtained on fibroblasts and PAECs of cloned piglet confirmed the donor cell lines CD39 expression detected by IHC. This was not the case with CD55 expression since the positivity detected by IHC was not confirmed with FACS and NB analyses. Conclusion: IHC is the method of choice when few cells for each clone are available, being more accurate than RT-PCR. Nevertheless, since this technique is not accurately quantitative, it needs to be complemented with alternative methods (Western Blotting or Real time PCR) to obtain a more complete evaluation of the expression pattern of the transgene
Perota, A., Brunetti, D., Charreau, B., Chatelais, M., Lagutina, I., Lazzari, G., Lucchini, F., Galli, C., Analysis of transgene expression in transfected somatic pig cell to be used as donor in nuclear transfer, Abstract de <<Joint Meeting of theInternational Pancreas and Islet TransplantAssociation (IPITA) and the InternationalXenotransplantation Association (IXA)>>, (Venezia, 12-16 October 2009 ), <<XENOTRANSPLANTATION>>, 2009; 16 (5): 436-436 [http://hdl.handle.net/10807/29360]
Analysis of transgene expression in transfected somatic pig cell to be used as donor in nuclear transfer
Lucchini, Franco;Galli, Cesare
2009
Abstract
Background: Somatic cell nuclear transfer is the method of choice to generate transgenic large animals. Success largely depends on a high expression level of the target protein. Selection of cell clones with the desired expression level is of paramount importance before nuclear transfer. In this work we compare different methods that can be used on a small number of cells and their predictive value. Methods: Two expression vectors for CD55 and CD39 were separately transfected to PK15 cell line. Following selection, five of the best growing clones resulting from each transfection were expanded and subjected to RT-PCR and Immunohistochemistry (IHC) analyses. For IHC analyses the mAB IA10 (BD-Pharmingen) – for CD55 – and the mAB BU61 (Ancell) – for CD39 – were used. The same antibodies were also used in Western blot (WB) analyses performed on samples subjected to non-reducing SDS–PAGE and electroblotted on PVDF membrane. The presence of the target transcripts was confirmed by Northern blot (NB) analyses using DIGlabeled probes (Roche). The proteins expression was also analysed by FACS conducted on chosen clones. Fibroblasts and PAECs deriving from one CD55–CD39 stillborn cloned piglet were subjected to IHC, NB and FACS analyses. Results: Three out of five (#24, #2 and #15) PK15–CD55 clones were positive to RT-PCR but only clone #24 was positive to IHC. Clone #24 was further analysed by NB, WB and FACS that confirmed the high expression level. Clone #2 revealed a low expression level by FACS not detected by IHC. All five PK15–CD39 clones were positive to IHC and RT-PCR analysis. Clone #10 was further analysed and confirmed positive by NB and WB. The IHC, NB and FACS data obtained on fibroblasts and PAECs of cloned piglet confirmed the donor cell lines CD39 expression detected by IHC. This was not the case with CD55 expression since the positivity detected by IHC was not confirmed with FACS and NB analyses. Conclusion: IHC is the method of choice when few cells for each clone are available, being more accurate than RT-PCR. Nevertheless, since this technique is not accurately quantitative, it needs to be complemented with alternative methods (Western Blotting or Real time PCR) to obtain a more complete evaluation of the expression pattern of the transgene
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