Construction of phosphodiesterase 4D homozygous knockout mice / 安徽医科大学学报

Objective @#The CRISPR / Cas9 technology was applied to construct PDE4D homozygous knockout mice to provide a basis for in-depth investigation of PDE4D gene function and mechanism of action．@*Methods@#A vector was constructed for PDE4D gene exon 4，5 microinjected into fertilized eggs of C57BL /6J mice，and PDE4D －/ - mice were obtained after maternal breeding and offspring mating，and the mice genotypes were determined by PCR product sequencing and genotype identification techniques．Changes in morphology and function of the major organs of the mice were detected using an ultrasound imaging system and H＆E staining，and the expression of PDE4D protein in the mice was verified by Western blot assay. @*Results @#The PDE4D －/ － mouse genotype was stably inherited， the mice were small，and there were no obvious morphological and histological changes in the major organs in vivo. The PDE4D expression was reduced or largely absent in the major tissues of PDE4D heterozygous or pure knockout mice，and the knockout effect was better．@*Conclusion @#PDE4D －/ － mice were successfully established using CRISPR / Cas9 technology，and no significant physiological abnormalities were found，which could be used for disease pathogenesis and drug research using PDE4D as the target.

Construction of THP-1 cell line with GPR108 gene deletion by CRISPR / Cas9 system and exploration of its function / 安徽医科大学学报

Objective@#To establish a Tohoku hospital pediatrics-1 (THP-1) cell line with G protein-coupled recep- tor108 ( GPR108) deletion and explore its functions．@*Methods@#According to the requirements of the clustered regularly interspaced short palindromic repeats ( CRISPR) / CRISPR-associated protein 9 ( Cas9 ) system ，two single guide RNAs (sgRNA1 and sgRNA2) paring to the flanking fragments of human GPR108 gene were designed and synthesized．The two oligonucleotides were inserted in the pL-CRISPR. EFS．GFP vector to generate the new recombinant vectors ( pL-CRISPR. EFS．GFP-sgRNA1 and pL-CRISPR. EFS．GFP-sgRNA2 ) ．The recombinant vectors and packaging plasmids (pMD2. G and psPAX2) ，were co-transfected into 293T cells to generate virus for infecting THP-1 cells．The GFP + cells were screened and isolated in 96-well culture plates by flow cytometry to obtain single-cell clones．PCR and Western blot were used to detect whether GPR108 was successfully knocked out in THP- 1 cells．Both GPR108 + / + and GPR108 －/ － THP-1 cells were treated with lipopolysaccharide (LPS) ．Interleukin 8 (IL-8) derived from the THP-1 cells，which were treated by LPS，was detected with Western blot and cytometric bead array ( CBA) analysis. @*Results@#The recombinant lentiviral vector pL-CRISPR. EFS．GFP-sgRNA was successfully constructed and single-cell clone F9 was obtained by flow cytometric sorting after transfection of THP-1 cells．PCR and Western blot both confirmed that F9 was a GPR108 －/ － THP-1 single-cell clone． LPS stimulated GPR108 －/ － and GPR108 + / + THP-1 cells，both Western blot and CBA results showed a significant decrease in IL- 8 synthesis and secretion in GPR108 －/ － THP-1 cells．@*Conclusion @#The GPR108 －/ － THP-1 cell clone is success- fully obtained based on the CRISPR / Cas9 system．GPR108 deletion in THP-1 cells treated by LPS leads to a decrease of IL-8 expression and secretion．It lays the foundation for further research on the molecular mechanisms of GPR108 in the immune inflammatory response.

Optimization of culture method of mouse primary hippocampal neurons and construction of HT22-GRK2 －/ － cells / 安徽医科大学学报

Objective@#To explore and optimize the primary culture method of neonatal mouse hippocampal neurons in vitro．To construct a G-protein-coupled receptor kinase 2 ( GRK2) knockout HT22 cell line．@*Methods @#Hippocampal tissue of C57BL6 /J mice on day 1－2 was taken，digested with trypsin and pipetted to form a cell suspension，and supplement was added to Neurobasal-A medium to maintain cell growth． CRSIPR / Cas9 gene editing technique was used to construct HT22-GRK2 －/ － cell line，and the knockout efficiency of GRK2 was detected by immunofluorescence staining and Western blot. @*Results @#Primary hippocampal neurons of newborn mice were put into six-well plates with 3 × 107 /well using a serum-free culture method，which could get a high purity and good activity ; HT22-GRK2 －/ － cell line was constructed successfully．@*Conclusion@#The primary culture method of mouse hippocampal neurons was successfully established and optimized，and HT22-GRK2 －/ － cell line was successfully constructed by CRSIPR / Cas9 gene editing technique.