Identification of differentially expressed genes of primary spermatocyte against round spermatid isolated from human testis using the laser capture microdissection technique

(整期优先)网络出版时间:2004-06-16
/ 1
Themethodoflasercapturemicrodissection(LCM)combinedwithsuppressivesubtractivehybridization(SSH)wasdevelopedtoisolatespecificgermcellsfromhumantestissectionsandtoidentifythegenesexpressedduringdifferentiationanddevelopment.Inthepresentstudy,over10,000primaryspermatocytesandroundspermatidcellsweresuccessfullyisolatedbyLCM.UsingthecDNAsfromprimaryspermatocytesandroundspermatids,SSHcDNAslibraryofprimaryspermatocyte-specificwasconstructed.TheaverageinsertsizeofthecDNAisolatedfrom75randomlypickedwhitecloneswas500bp,rangingfrom250bpto1.7kb.Usingthedot-blotmethod,atotalof421cloneswereexamined,resultingintheidentificationof390positiveclonesemittingstrongsignals.PartialsequenceofcDNAspreparedfromeachclonewasdeterminedwithanoverallsuccessrateof84.4%.GenesencodingcytochromecoxidaseⅡandtherescuefactor-humaninweremostfrequentlyexpressedinprimaryspermatocytes,suggestingtheirrolesinvolvedinmeiosis.