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Construction and Functional Verification of siRNA Eukaryotic Expression Vector Directed at the Follicular Inhibin Alpha Gene in Ye Mule Sheep

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*

1College of Agriculture, Ningxia University, Yinchuan, 750021, China
2College of Animal Science and Technology, Shihezi University, Shihezi, 832000, China 
3Kazakh National Agrarian University, Almaty, Abay Avenue 8, Kazakstan
4S Seifullin Kazakh Agro Technical University, Nur Sultan Probed Avenue, 62, Kazakstan

*      Corresponding author: 1508217366@qq.com; xndaxue@126.com

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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.

Fig. 4.

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.

Fig. 5.

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.

Fig. 6.

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.

Pakistan Journal of Zoology

October

Vol. 52, Iss. 5, Pages 1631-2026

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