Rnai Technology To Inhibit The Human Znf313 Gene Expression Genetic Polymorphism With Alzheimer's Disease Association Studies

Spermatogenesis is a complex cell differentiation process that results in a series of dramatic molecular and morphological changes in male germ cells. This process requires the highly regulated expression of a number of genes. Studies of male idiopathic infertility have found that many genes both on sex chromosomes and on autosomes are requred for normal spermatogenesis. Further studies on these genes and their functions would help us to understand the molecular mechanism of spermatogenesis, as well as to clarify the genetic pathology of azoospermia.In recent years our team has devoted itself to the cloning and functional study of the genes related to male infertility. Most of the spermato-genesis-related genes that we have cloned are classified as zinc finger proteins, for instance ZNF230, ZNF463, and ZNF313. Human ZNF313 gene, which is isolated by mRNA differential display between the testes of fertile adults andazoospermic patients, is localized at 20ql3 and encodes a 228aa-long zinc finger protein, which contains both ring finger and C2H2 domain. Two transcripts of ZNF313 gene are produced via differential splicing. The transcripts are expressed in multiple tissues, while the shorter 750bp message is highly expressed in testis.In order to investigate the feasibility of RNAi targeted human ZNF313 gene, we constructed five short hairpin RNA recombinant expression vectors targeted the human ZNF313 gene. The vectors contained HI and U6 promotor respectively. After HL-7702 cells, a human normal liver cell line, were transfected with the five constructs, semi-quantitative RT-PCR and western blot were applied to detect the gene's expression. We found that all the five constructs can knock-down the ZA/F313 gene's expression and the reductions of mRNA levels were different from each other. The construct pSUPER*insert3 reduced the expression of ZNF313 to the least amount. MTT assay suggested that RNAi-induced low-expression of ZNFi 13 gene might not affect the cell proliferation.In conclusion, recombinant shRNA expression vectors could induce the RNAi in cultured HL-7702 cells and efficiently knock down the expression of ZNF313 gene. This result established a solid basis for the further functional analysis of this gene via RNAi technique.