Construction and Functional Verification of siRNA Eukaryotic Expression Vector Directed at the Follicular Inhibin Alpha Gene in Ye Mule Sheep
Zengwen Huang1,2, WuReliHazi Hazihan2*, Baheti Bodai2, Kadyken Rizabek3, Nuralieva Ulzhan3, Omarova Karlygash4, Juan Zhang1 and Yaling Gu1*
Agarose electrophoresis of total RNA.
Lanes 1, 2, 3 total RNA from three representative YM sheep ovarian tissue extracts. Migration of 5S, 18S and 28S ribosomal RNA bands is indicated. The ratio of A260/A280 of 1.8.
Amplification of a cDNA fragment encoding the INh α gene from YM sheep.
M, DL5000 DNA Marker; Lanes 1 and 2, The amplification product of INH a gene.
The recombinant plasmid pGenesil10-3 p – siRNA was identified by enzyme digestion.
M, DL5000 DNA Marker; Lanes 1, pGenesil10-3 p–siRNA plasmid not digested by enzyme; Lanes 2 and 3, Identification of recombinant plasmid pGenesil10-3 p– siRNA by PstI enzyme digestion.
Granulosa cells growing for different times.
A, after 24 h of granulosa cell culture, adherent growth entered into hysteresis period; B, granulosa cell culture for 48 h and entered the logarithmic phase; C, granulosa cell entered a stable phase after 72 h of culture; D, granulosa cell were culture for 96 h and entered the decline period.
Experimental diagram of plasmid transfection granule cells.
A, granulosa cell not transfected with plasmids in logarithmic phase; B, results of pgenesil 10-3p-HK plasmid transfected granulosa cells at logarithmic growth stage; C, results of pgenesil 10-3p-HK plasmid transfected granular cells at logarithmic growth stage.
Three picture show the expression of INHα gene in granulosa cells after interference.
A, the interference results were identified by Q-PCR; B, protein expression results of INHα gene in granulosa cells; C, interference results of western bolt identification.