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Objective To establish RNase L gene knockout HEK 293 cell lines using CRISPR/Cas9 system.Methods Small guide RNA ( sgRNA) sequences of human RNase L were designed and sgRNAs were inserted into pCas-Guide and pCas-guide RNA(gRNA) vectors were obtained.The donor DNA sequences of the homologous arm were designed for RNase L knockout .In the presence of the right homologous arm , the resistance gene of hygromycin B and the left homologous arm as templates of homology-directed repair , the donor DNA template was amplified by overlopping PCR and cloned into the pBackZero-T expression vector and pBackZero-T-RNase LK vector was obtained .The pCas-gRNA vector and pBackZero-T-RNase LK vector were co-transfected into HEK293 cells to establish the stable expression cell line of RNase L gene knockout .Cells were cultured with hygromycin B , while Western blotting and DNA sequencing were used to analyze the gene of RNase L knockout from genome .Results and Conclusion The pCas-gRNA vector and pBackZero-T-RNase LK vector were successfully constructed.Five RNase L gene knockout HEK293 cell lines were generated,contributing to the study of the biological function and molecular mechanism of RNase L .
RESUMEN
Objective To explore the role of inositol requiring enzyme 1 α (IRE1α) mediated endoplasmic reticulum stress associated apoptotic molecules in hippocampal neuronal injury in rats with status epilepsy following lithium-pilocarpine.Methods All 96 Wistar rats were randomly divided into control group and status epilepsy (SE) group.The SE group was further divided into 5 subgroups (3,6,12,24,48 h) according to different time points.pmmunofluorescence was used to observe the expressions of endoplasmic reticulum stress (ERS) markers glucose-regulating protein 78 kd (GRP78) and phosphoIRE1α (active form of endoplasmic reticulum resident protein IRE1α) at the CA3 area of rats in each group.Then,the expressions of IRElα mediated downstream apoptotie markers phospho-c-JunN-terminalkinase (JNK) and caspase12 were detected.Finally,TUNEL assay was used to observe neuronal apoptosis of hippocampal CA3 area at different time points after SE in rats.Results Immunofluorescence showed that GRP78 and phospho-IRE1α positive neurons were significantly increased in the SE subgroups compared with control group (6.90％ ± 0.96％,4.60％ ± 1.12％,respectively) and 12 h subgroup reached the peak (GRP78:87.45％ ±3.63％,F =356.82,P ＜0.05; phospho-IRE1α:86.90％ ±3.82％,F =300.80,P ＜ 0.05).Immunohistochemistry and Western blot demonstrated that the levels of phospho-JNK and caspase12 in the SE subgroups were significantly higher than that in the control group which reached the peak at 12 h after SE.The changes were in accord with phospho-IRE1α.Simultaneously,hippocampal neuronal apoptosis was detected in each SE subgroup and was most severe at 12 h after SE,which showed similar changes to the expressions of phospho-IRE1α,phospho-JNK and caspase12.Conclusions ERS was induced in rats following SE evidenced by increasing the expression of GRP78.IRE1α may promote hippocampal neuron apoptosis in rats following SE through activating JNK and caspase12.