〔Abstract〕 Objective To explore and optimize the primary culture method of neonatal mouse hippocampal neuronsin 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 ofGRK2was 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.