ABSTRACT
Objective To establish a method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9, and analyze its off-target effect. Methods The gene editing site for MAD2L1 gene was designed by CHOP?CHOP, and the Cas9?MAD2L1 vector was constructed based on the designed editing site. Cas9?MAD2L1 was then transfected into NIH/3T3 cells and screened with puromycin, followed by observing GFP expression using fluorescence microscopy. The genomic DNA from transfected cells was extracted and a partial fragment of MAD2L1 gene was amplified by PCR. T7E1 analy?sis and Sangger sequencing were used for gene editing and off?target analysis. Results After Cas9?MAD2L1 transfection and puromycin screening, a large number of GFP?expressing cells were observed under the fluorescence microscope. Combined the PCR result with TE71 analysis, the amplified 228 bp PCR products can be digested into 166 bp and 62 bp fragments. The se?quencing result showed that the second exon of MAD2L1 gene was successfully edited, and the off?target effect was undetected in our system. Conclusions The method for specific gene editing of the second exon of mouse MAD2L1 gene by CRISPR/Cas9 is successfully established, and off?target effect of MAD2L1 gene is not detected.
ABSTRACT
Ventricular remodeling is one of the main causes of heart failure. A large number of studies have shown that inflammation plays an important role in the occurrence and development of ventricular remodeling. Recent studies found that chronic inflammation mediated by T cells is closely related to the progression of ventricular remodeling. This review summarized the recent research progress of T lymphocyte subsets in ventricular remodeling.
ABSTRACT
Ventricular remodeling is one of the main causes of heart failure. A large number of studies have shown that inflammation plays an important role in the occurrence and development of ventricular remodeling. Recent studies found that chronic inflammation mediated by T cells is closely related to the progression of ventricular remodeling. This review summarized the recent research progress of T lymphocyte subsets in ventricular remodeling.
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Purpose To investigate the influence of long chain non-coding RNA (lncRNA)-HOTAIR on endometrial cancer cell proliferation,invasion,metastasis and other biological behaviour.Methods 20 cases of endometrial carcinoma tissue specimen,20 cases of hyperplasia tissue sample and 10 cases of normal tissue specimen were collected.Difference expression of lncRNA-HOTAIR in normal endometrium,hyperplasia endometrium tissue and endometrial carcinoma tissue at all periods were detected with RT-PCR assay.HOTAIR-siRNA transfection into Ishikawa cells was utilized with Lipofectamine 2000.MTT experiment was used to detected the proliferation ability of cells in all groups.Transwell chamber experiment was used to test the migration and invasion ability of cells in all groups.Results The gene expression level of of lncRNA-HOTAIR in endometrial carcinoma group at all stages was prominently increased compared with normal endometrium group (P ＜ 0.05).The expression level of lncRNA-HOTAIR in simple hyperplasia endometrium group and atypical hyperplasia endometrial group was not significantly different (P ＞ 0.05).Cell proliferation,invasion ability and migration ability of HOTAIR-siRNA targeting suppression group were lower than the blank control group and the negative control group significantly (P ＜ 0.05).Conclusion lncRNA-HOTAIR may involved in occurrence and development of endometrial cancer,which may play an important role in the aggression and metastasis of endometrial cancer.