Objective To clone the full-length of human Gill cDNA and construct the recombinant lentiviral expressing vector pLOX-Gfil for eukaryotic expression,providing a basis for further study on the biological functions of Gfil.Methods Total RNA was isolated from K562 cells,and the full-length Gfil cDNA was amplified by RT-PCR and then ligated with pGEM-T vector after retrieve and purification.The ligation product was transformed into competent cells DH5a.The positive recombinant clones were selected and identified by a complementation,restriction endonuclease digestion.The cloning vector and the lentiviral vector pLOX first digested with BarnH I were ligated and transformed.The enzyme and PCR analyses were performed to confirm the recombinant vector,and then DNA sequence analysis.Results A fragment of 1.2 kb was obtained by RT-PCR.The enzyme and PCR analyses revealed that the correct Gfil cDNA was cloned.The sequence of cloned cDNA was identical to the sequence deposited in GenBank (NM005263).Conclusion Gfil was cloned correctly and the recombinant lentiviral vector pLOX-Gfil for eukaryotic expression was constructed successfully.

Triptolide is a key active component of the widely used traditional Chinese herb medicine Tripterygium wilfordii Hook.F.Although triptolide exerts multiple biological activities and shows promising efficacy in treating inflammatory-related diseases,its well-known safety issues,especially reproductive toxicity has aroused concerns.However,a comprehensive dissection of triptolide-associated testicular toxicity at single cell resolution is still lacking.Here,we observed testicular toxicity after 14 days of triptolide exposure,and then constructed a single-cell transcriptome map of 59,127 cells in mouse testes upon triptolide-treatment.We identified triptolide-associated shared and cell-type specific differentially expressed genes,enriched pathways,and ligand-receptor pairs in different cell types of mouse testes.In addition to the loss of germ cells,our results revealed increased macrophages and the inflammatory response in triptolide-treated mouse testes,suggesting a critical role of inflammation in triptolide-induced testicular injury.We also found increased reactive oxygen species(ROS)signaling and down-regulated pathways associated with spermatid development in somatic cells,especially Leydig and Sertoli cells,in triptolide-treated mice,indicating that dysregulation of these signaling pathways may contribute to triptolide-induced testicular toxicity.Overall,our high-resolution single-cell landscape offers comprehensive information regarding triptolide-associated gene expression profiles in major cell types of mouse testes at single cell resolution,providing an invaluable resource for understanding the underlying mechanism of triptolide-associated testicular injury and additional discoveries of therapeutic targets of triptolide-induced male reproductive toxicity.