1.Construction of H22 GP73 knockout gene stable strain using CRISPR/Cas9 gene editing system and identification of functions
Jiankang CHEN ; Congwen WEI ; Hui LIANG ; Beihan WANG ; Hui ZHONG ; Xiaoli YANG
Military Medical Sciences 2016;40(7):549-553
Objective To knock out the GP73 gene in H22 cells originating in mice using CRISPR/Cas9 gene editing system and construct H22 GP73 gene knockout stable strain for identification of its functions .Methods Two pairs of sgRNAs that could specifically identify the upstream and downstream of GP 73 gene first promoter were designed before a recombinant eukaryotic expressional plasmid was constructed using pX 459 .After enzyme digestion and sequencing , two pairs of recombinant plasmids were co-transfected into H22 cells before puromycin was used to screen positive cells and monoclonal cells which stably knocked out GP 73 gene were developed .The knockout effect was measured by Western blotting.Cell Titer 96? AQueous One Solution Assay was used to detect the effect on cell reproductive capacity when the GP73 was knocked out .The transferability was detected through wound healing test .Results The result of Western blotting suggested that GP73 protein was undetected in the construction of H22 GP73 knockout gene stable strain after transfection.The transfer and reproduction slowed down .Conclusion H22 GP73 gene knockout stable strain can be successfully built using CRISPR/Cas9 gene editing system ,thus facilitating studies on the function of GP 73 in hepatocarcinogenesis .
2. The establishment of a chyle testing method based on n-hexane extraction system
Beihan WANG ; Ruiling WANG ; Xiaoli ZHANG ; Cui WANG ; Qi DING ; Liping LIU ; Rong ZHAO ; Xiaoli YANG
Chinese Journal of Laboratory Medicine 2018;41(7):527-530
Objective:
To set up a chyle (n-hexane) testing method based on n-hexane extraction system.
Methods:
The hydrothorax specimen which was positive in chyle testing was selected and the feasibility was evaluated by replace Diethyl ether with n-hexane. The optimal extraction volume ratio for the new chyle testing was confirmed and sultan Ⅲ dye process was improved. Then, the new chyle testing method and its standard operation process was established.A total of 120 specimens with triglyceride(TG)in different levels were collected and divided into three groups with 40 specimens for each group (group A. TG≤1.7 mmol/L; B. 5.6