Expression inhibitor of porcine hdac2 gene and its application
A technology of gene expression and inhibitors, applied in the biological field, can solve the problems of low production efficiency and limit the promotion and application of somatic cell cloned pigs, and achieve the effects of reducing negative effects, increasing the number of cells in blastocysts, and increasing the rate of blastocysts
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Embodiment 1
[0035] 1. Design and synthesis of porcine HDAC2 gene and siRNA of porcine HDAC2 gene
[0036] According to the porcine HDAC2 gene sequence (sequence number: XM_001925318.6), referring to the principle of siRNA design, three pairs of siRNA were designed and synthesized by Shanghai Gemma Pharmaceutical Technology Co., Ltd. with the corresponding target sites of the porcine HDAC2 gene, respectively denoted as HDAC2-siRNA1, The sequences of HDAC2-siRNA2 and HDAC2-siRNA3 are shown in Table 1 below:
[0037] Table 1 siRNA sequence
[0038]
[0039] 2. The inhibitory effect of siRNA on genes
[0040] 2.1 siRNA solution preparation
[0041] Centrifuge the tubes containing the dry siRNA powder, and then add 150 μL of sterile, DNase & RNase-free embryo injection-grade water to each tube (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.). Concentration of 20mM, cap the bottle tightly and shake to dissolve. After centrifugation, dispense 5 μL / tube into sterile, DNase & RNas...
Embodiment 2
[0064] 1. The effect of CAY10683 on the developmental performance of pig somatic cell cloned embryos
[0065]The small molecule CAY10683, which specifically inhibits HDAC2, was added to the culture medium to incubate and culture the reconstituted embryos of pig clones at the 1cell stage for 24 hours, and then replaced with normal medium to continue the culture. The NC group was supplemented with the same amount of untreated normal medium. The cleavage rate of cloned and reconstituted embryos in the experimental group was observed on day 2 and the blastocyst development rate on day 7, and nuclear fluorescent staining was performed on blastocyst cells, and the total number of cells was counted. The results are shown in Table 3.
[0066] Table 3 Effects of different concentrations of CAY10683 on the developmental performance of pig somatic cell cloned embryos
[0067]
[0068] As can be seen from the results in Table 3, compared with the NC group, the CAY10683 treatment grou...
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