Construction Of FAAP Knockout Mice And Preliminary Phenotypic Analysis

Focal adhesion associated protein(FAAP),encoded by murine D10wsu52e gene, is about55kD protein. It is a highly conserved protein and widely expressed in bacteria, archaea andmetazoan. The expression analysis in different mice tissues found that FAAP is highly expressedin reproduction organs. We speculated that it was a gene to closely related the reproductivesystem. To explore the FAAP function in male reproductive system, we studied FAAPexpression patterns in male reproductive system using reverse transcription PCR and WesternBlot. FAAP m RNA expressed in testis, epididymis, vas deferens and prostate, but not found inthe seminal vesicle, and it is highly expressed in testis and epididymis. Western Blot FAAPprotein expression quantity in epididymis and testis of male C57BL/6j mice after the birth of1,7,14,21,28,35,42days found, the expression of FAAP protein is gradually increased with thedays after birth. It suggests that FAAP may play a important role in morpHological development,maintain the function and sperm maturation of mice epididymis. The above results can beconsidered control groups to observe FAAP expression changes in different development periodof epididymal and testicular in knockout group.We use gene targeting technology to build FAAP knockout mice-KO (knockout) First mice.KO First is a multipurpose model, similar to conditional knockout strategy, on both sides of thetarget fragment loxP loci were placed in the same direction. At the same time, a SA-IRES-reporter flanked with FRT sites fragments were placed in5’intron of target segments. Using thetarget gene promoter expression report (reporter) we can test the expression of interest genethrough test report gene expressions. And mice expressing Flp can remove SA-IRES-reporterfragments, get the regular flox mice. KO First flox mice is different from conditional KO floxmice, the pHenotype of conditional KO flox mice is normal, but KO First flox mice is KOpHenotype. We can detect pHenotype of KO-First mice to be the preliminary pHenotypeanalysis of FAAP KO mice. The knockout mice we used in this study is KO-First mice whichinsert the FRT-lacZneo-FRT-loxP components and loxP loci, with LacZ reporter gene. LacZstaining can position and test expression of FAAP.According to ours results, FAAP highly expressed in the brain and testis epididymis in theKO-First mice. Comparing fertility in KO-First mice and C57BL/6J mice found thatconception rate of KO–First mice is significantly decreased.And offspring of KO-First mice began to die from about14days after birth, and the mortality rate is very high between28daysafter birth and42days. These results suggest that FAAP play an important role in the process ofits growth and development. FAAP KO First mice mating with Flper mice, remove FRT-LacZneo-FRT components, can get regular FAAP flox mice. FAAP flox mice mating withDefb41iCre/wt can obtain FAAP conditional knockout mice specificity expressed in epididymis.KO-First knockout mice model can be used to undertake FAAP knockout micepreliminary pHenotypic analysis, make preparation for detecting the expression of FAAP in theinitial period of epididymis, detect FAAP influence on sperm maturation and FAAP detection inthe role of the male reproductive system in mice, research and development new birth controlpills and make the theoretical basis for treatment of male infertility.