Establishment Of Fank1 Knockdown Transgenic Mouse Model And The Reproductive Function Of The Gene In Male Mice

ObjectiveFankl, a testis-specific expression gene, expressed extensively from mitosis to haploid in spermatogenesis. The studying on the characters and the functions of Fankl have important roles in discovering the mechanisms of spermatogenesis, preventing the male infertility caused by aberrant development of sperm, genetic diagnosis and the exploiture of pharmaceutical contraceptives. Therefore, we established a mouse model of infertility using Fankl gene knockdown by RNA interference, and carried out a set of analysis on reproductive function and phenotype of infertile male mice to discover the important role of Fankl in spermatogenesis and male infertility.Materials and MethodsMaterials1. Human embryo kidney cell line 293T2. SPF mice C57BL/6, B6D2F1 and ICR3. Reagents for Cell culture and cell transfection4. Reagents for Molecular cloning and PCR experimental5. Reagents for RT-PCR, Real-time-PCR and microRNA extraction6. Reagents for Southern blot and Western blotMethods1. Construction of RNA interference plasmids targeting for Fankl and detection of its expression on cellular level.(1) Construction of pSUPER-shFankl recombinant plasmid and pDsRed-Fankl fusion expression construct. According to Fankl cDNA gene sequences, we designed 2 interference sequences using http://www.dharmacon.com/online design software, cloned them into pSUPER-neo-GFP expression vector with H1 promoter. Fankl cDNA sequences was amplified from testicular tissue of adult C57BL/6 mice recombinant pDsRed-Fankl plasmid was constructed.(2) Cotransfection of pSUPER-shFankl and pDsRed-Fankl were transfected into 293T cells to detect the interference effect on Fankl using fluorescence detection, RT-PCR and Western blot.2. Establishment of Fankl gene knockdown mice model(1) Plasmids with of high interference efficiency were selected and cut by a single enzyme, purified with gel extraction kit, and then the fragment was injected into fertilized eggs of BDF1 mice through microscopic injection. the fertilized eggs with exogenous gene were transplanted into oviduct of pseudopregnant females mice, positive new born mice were detected with PCR and Southern blot.(2) Breeding of Fankl knockdown trans genic mice3. Phenotype analysis of Fankl knockdown transgenic mice(1) Extracted microRNA from Fankl knockdown transgenic mice testis, detected Fankl siRNA expression with RT-PCR.(2) Detected Fankl expression in Fankl knockdown transgenic mice with RT-PCR, Real time-PCR and Western blot.(3) Testis weight and sperm counts of Fankl knockdown transgenic mice and wild type mice were examined.(4) Fankl gene knockdown mice were mated with wild mice to observe litter size and litter interval. Results1. PCR analysis showed that the recombinant plasmid pSUPER-shFankl 631#, pUPER-shFankl 247# and pDsRed-Fankl expression construct were constructed successfully, and DNA sequence was identified by sequcing analysis.2. Fluorescence microscope examination showed that shFankl 631# and 247# can inhibit red fluorescent protein expression, Inhibition efficiency is 90% and 95% respectively in 36 h after 293T cells were transfected. The results of RT-PCR and Western blot suggested that Fankl gene was inhibited significantly at mRNA and protein level by Fankl siRNA (p<0.05).3. We Injected pSUPER-shFankl 631# into prenuclear by fertilized eggs of BDF1 mice by micrinjection. Among 285 embryos,203 embryos were alive. We transplanted the living fertilized eggs into oviduct of 8 ICR pseudopregnant female mice. There were 14 founders from 53 pups with PCR and Southern blot analysis detected and the positive rate was 26.4%.4. Fankl siRNA was expressed in testicular tissue of 7 Fankl knockdown transgenic mice. RT-PCR suggested Fankl mRNA expression in testicular tissue of Fankl knockdown transgenic mice was significantly reduced compared with that of the normal mice. To confirm the results, we used Real Time-PCR and Western blot to detect Fankl gene expression in Fankl knockdown transgenic mice, the results showed that Fankl gene expression in 7 Fankl knockdown transgenic mice were decreased compared with that of wild type mice. And in F01, F02, F011, F045 transgenic mice Fankl gene showed notably down-regulated expression.5. Testis of adult male Fankl knockdown transgenic mice, compared with wild type mice, Statistical analysis suggested that the testicular tissue weight of Fankl knock down transgenic mice and wild type mice has non-significance difference, p>0.05.6. By statistical analysis, the sperm count of Fankl knockdown transgenic mice showed significant difference with that of wild mice (p<0.05), litter size and litter interval of Fankl knockdown transgenic mice had significant difference with that of wild mice.Conclusion1. Constructed the RNA interference construction of testis-specific gene-Fankl in mouse and the construction of pDsRed-Fankl. At the cell level, it showed the interference effects by the RNA interference construction of Fankl.2. Established Fankl knockdown transgenic mouse model successfully.3. Fankl is an important factor in the transcription regulation, in the transgenic mouse the expression of Fankl was inhibited, it caused spermatogenesis deficiency.