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| Construction of RNA Interference Vector of Chicken (Gallus domesticus) FGF8 Gene and Its Effect on the Formation of PGCs |
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Abstract Abstract As early precursors for sperm cells, primordial germ cells (PGCs) are widely used to study spermatogenesis. Fibroblast growth factors (FGFs) are essential in regulating the formation of PGCs. In order to investigate the regulation of FGF8 on the formation of PGCs, we established a chicken embryonic stem cell line with FGF8 low expression, through constructing a small hairpin RNA (shRNA) lentivirus vector for chicken (Gallus domesticus) FGF8, and further investigate the role that FGF8 plays in the differentiation of male germ cells. Three shRNA interfering vectors targeting chicken FGF8 were constructed and transfected into chicken DF1 cells transiently. qRT-PCR was used to detect the interference efficiency of different interference targets on FGF8, and the shRNA vector with the best interference efficiency was packaged in lentivirus. The induction of retinoic acid (RA) was performed in an ES cell lines knocked down by FGF8, and the morphological changes of the cells in each group were observed, also, the cells of each group were collected on day 0, 2, 4 and 6, respectively. The expression of related reproductive genes, like Nanog, Oct4, Cvh, C-kit, Blimp1 and so on, and the efficiency of PGCs formation were detected by qRT-PCR, flow cytometry, and immunofluorescence. The results showed that 3 lentiviral interference vectors and 1 negative vector for FGF8 were successfully constructed and named as shFGF8-1, shFGF8-2, shFGF8-3, shNC. By transfecting DF1 cells, shFGF8-2 had been found the highest interference efficiency relative to shFGF8-1 and shFGF8-3. Hence, shFGF8-2 was packaged with lentivirus and the titer was 5×108 TU/mL, the interference efficiency was (70±4.31)% in ESCs. For ESCs with low expression of FGF8, we found that under normal RA-induced for 4 d, PGC-like cells were significantly increased, and the pluripotency associated gene Nanog, was significantly down regulated (P＜0.01). Meanwhile, the germ cell marker gene Cvh, and C-kit, were significantly up-regulated (P＜0.01). Moreover, the results of immunofluorescence and flow cytometry further confirmed that FGF8 knockdown could induce significant excess of CVH+PGCs (P＜0.01), compared to the control group after 4 d of induction. Collectively, a FGF8-specific lentivirus vector stably transfected with chicken embryo stem cells was successfully constructed, and a novel function of FGF8 in the formation of PGCs in vitro was covered.
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Received: 21 March 2018
Published: 06 August 2018
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| Fund:The National Natural Science Foundation of China;The National Natural Science Foundation of China |
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