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| The Preparation and Infection Efficiency Analysis of the Recombinant Adenovirus Expressing Chicken (Gallus gallus) miR-17-92 Cluster |
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Abstract Abstract The miR-17-92 cluster plays crucial roles in cell proliferation, differentiation, apoptosis, animal development and tumorigenesis in mammals. However, the roles and mechanisms of miR-17-92 cluster in chicken growth and development remain to be elucidated. To explore the roles and the mechanisms of miR-17-92 cluster in chicken growth and development, in this study, chicken miR-17-92 cluster was PCR amplified from chicken genomic DNA and ligated into pacAd5 miR-GFP Shuttle Vector to yield recombinant vector containing the chicken miR-17-92 cluster. The resultant vector pacAd5-miR-17-92 cluster and backbone plasmid pacAd5 9.2-100 were respectively linearized with PacⅠ, and then cotransfected into AD293 cells to generate the recombinant adenovirus expressing miR-17-92 cluster, named as rAd5-miR-17-92 cluster, by homologous recombination. The virus titer was determined by using TCID50 assay after the virus amplification. The recombinant adenovirus rAd5-miR-17-92 cluster was identified by PCR and sequencing. The recombinant adenovirus rAd5-miR-17-92 cluster and empty vector virus were inoculated into the immortalized chicken preadipocytes (ICPA), respectively, and the expression of miR-17-92 cluster member was detected by Real-time stem-loop RT-PCR. The results showed that the titers of both of recombinant adenovirus rAd5-miR-17-92 cluster and empty vector adenovirus rAd-control reached 1×109 pfu/ml. PCR identification showed a specific product of 879 bp was amplified from the recombinant adenovirus containing chicken miR-17-92 cluster, and sequencing result showed that the acquired sequence was identical to the expected sequence. Real-time stem-loop RT-PCR analysis showed the relative expression levels of miR-19a and miR-20a, two members of miR-17-92 cluster, were significantly increased to (2.2±0.2)-fold in ICPA cells infected by recombinant adenovirus when compared with those in cells infected with empty vector virus (P＜0.01). Taken together, these results suggested the recombinant adenovirus rAd5-miR-17-92 cluster was successfully prepared. Four human embryonic kidney epithelial cells (HEK) (293A, 293T, AD293 and HEK293), ICPA cells, chicken fibroblast (DF1), human epithelial cells (HaCaT) and sheep embryonic fibroblasts (SEF) were individually infected by rAd5-miR-17-92 cluster, and infection efficiency was detected by fluorescence microscopy. The results showed that the infection efficiency of rAd5-miR-17-92 cluster was different in different cell types, it was high in 293A, 293T, AD293 and HEK293, moderate in HaCaT and SEF, but Low in ICPA and DF1 cells. Taken together, these data suggested that recombinant adenovirus expressing miR-17-92 cluster was successfully prepared, which can contribute to further study on the roles and mechanisms of miR-17-92 cluster in chicken growth and development.
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Received: 22 February 2017
Published: 06 August 2017
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| Fund:;the China Agriculture Research System |
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