Establishment, Growth, Proliferation, and Gene Expression of Buffalo (Bubalus bubalis) Transgenic Fetal Fibroblasts Containing Human Insulin Gene, and Production of Embryos by Handmade Cloning Using These Cells.
Cell Reprogram
; 20(2): 135-143, 2018 04.
Article
em En
| MEDLINE
| ID: mdl-29446977
ABSTRACT
The aim of the present study was to compare transgenic cells, containing human insulin gene kept under the control of mammary gland-specific buffalo beta-lactoglobulin promoter, and their counterparts, that is, nontransgenic cells, for examining their potential for the production of embryos following somatic cell nuclear transfer (SCNT). The gene construct was delivered into buffalo fetal fibroblasts (BFF) by nucleofection following which, the transfected cells were selected by culture in the presence of G418 for 3 weeks. Transgene integration into BFF genome was confirmed by polymerase chain reaction (PCR) and reverse transcriptase PCR. At passage 8-10, the growth rate, cell proliferation rate, and quantitative expression of certain genes were compared between transgenic and nontransgenic cells. The growth rate and cell proliferation rate was significantly lower (p < 0.05) for transgenic than for nontransgenic cells. Using quantitative real-time PCR it was found that the expression level of CASPASE 3, CASPASE 9, BAX, and P53 was significantly higher (p < 0.05) and that of HDAC1 and IGF-1R was significantly lower (p < 0.05) in transgenic compared with nontransgenic cells. The differences in the relative expression level of BCL-XL, MCL-1, DNMT1, DNMT3a, GDF9, FGF2, and G6PD between the two groups were not significant. Furthermore, when the two cell types were used as donor cells for production of embryos by handmade cloning, the blastocyst rate was significantly lower (p < 0.05) with transgenic (35.69% ± 1.78%) than with nontransgenic cells (48.75% ± 2.38%). In conclusion, these results indicate that differences were present between transgenic and nontransgenic cells, which may affect the efficiency of SCNT when used as donor cells.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Blastocisto
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Búfalos
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Clonagem de Organismos
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Técnicas de Transferência Nuclear
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Insulina
Limite:
Animals
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Humans
Idioma:
En
Revista:
Cell Reprogram
Ano de publicação:
2018
Tipo de documento:
Article
País de afiliação:
Índia