Publication: Fare embriyolarına mikroenjeksiyon yöntemi ile HBV DNA aktarılarak HBV replikasyonu ve gen ekspresyonunun gerçekleştiği transgenik fare modelleri oluşturulması
Abstract
Hepatit B Virüsü (HBV) akut, kronik karaciğer hastalıklarının yanısıra, hepatoselüler karsinomaya (HCC) yol açan bir DNA virüsüdür. İnsan sağlığını tehdit eden en önemli risk faktörlerinden biri olan HBV'nin konak sayısının sınırlı olması ve virüsün üretildiği in vivo kültür sisteminin yokluğu, virüs ile ilgili araştırmalarda HBV genomunu (kısmi/ bütün) taşıyan transgenik fare modellerinin önemini arttırmıştır. Bu çalışmada ülkemiz için de önemli bir sağlık sorunu yaratan kronik HBV enfeksiyonunun tedavisinde kullanılacak ilaç ve aşı çalışmalarının yanısıra, HCC ile olan ilişkisinin de araştırılabileceği transgenik fare modeli oluşturulması planlanmıştır. HBV genomunu taşıyan plazmitten saflaştırılan tüm HBV genomu mikroenjeksiyon yöntemi ile fertilizasyon sonrası elde edilen CD-1 soyu fare tek hücreli embriyolarına aktarılmıştır. HBV genomu taşıyan fare embriyoları anestezi altındaki pseudopregnant (yalancı gebe) CBGF1 soyu farelere yerleştirilmiştir. Manipülasyon sonrası doğan yavrulardan transgenik olanlar polimeraz zincir reaksiyonu (PZR) ve dot-blot yöntemi ile saptanmıştır. HBV gen ekspresyonu transgenik farelerin serumlarında ve karaciğerlerinde ELİSA ve immünohistokimyasal yöntemlerle HBV yüzey antijeni (HbsAg) saptanarak ve fare karaciğer dokularından elde edilen RNA'lara RT PZR uygulanarak belirlenmiş ve ülkemizde ilk kez HBV trnasgenik fare modeli gerçekleştirilmiştir.
TRANSGENIC MICE MODELS IN WHICH HBV REPLICATION AND GENE EXPRESSION OBTAINED BY MICRO ENJECTION METHOD The Hepatitis B virus (HBV) is a DNA virus that causes acut, chronic liver diseases and also leads to hepatocellular carcinoma (HCC). The limited host range of HBV and the inability to grow in vitro, have increased development of HBV transgenic mice models that carry whole/ partial HBV genome in the viral experiments. In this study, to develop transgenic mice model has been planned to determine the drug/ and vaccine in the threapy of chronic HBV infection and HBV/ HCC relation which is an important health problem in our country. The HBV genome was purified from the Pt HBV 1,3 plazmid with terminally redundant DNA construct (HBV 1,3) and transferred to the one-celled embriyos of CD-1 mouse strain which were obtained after fertilization by pronuclear microenjection. The mice embriyos were transferred to the pseudopregnant CBGF 1 mouse strain that were under anesthesia. Transgenic offspring which were born after manipulation have been determined by polimerase chain reaction and dot-blot hybridization assays. HBV gene expression of transgenic mice has been shown to determine hepatitis B virus surface antigen by ELISA and immunohistochemical methods in their sera and livers and RT PCR was applied to the RNA samples which have been obtained from liver specimens. In this sudy, production of first transgenic animal in Turkey which is the transgenic mice containing and expressing HBV genome, has been accomplished.
TRANSGENIC MICE MODELS IN WHICH HBV REPLICATION AND GENE EXPRESSION OBTAINED BY MICRO ENJECTION METHOD The Hepatitis B virus (HBV) is a DNA virus that causes acut, chronic liver diseases and also leads to hepatocellular carcinoma (HCC). The limited host range of HBV and the inability to grow in vitro, have increased development of HBV transgenic mice models that carry whole/ partial HBV genome in the viral experiments. In this study, to develop transgenic mice model has been planned to determine the drug/ and vaccine in the threapy of chronic HBV infection and HBV/ HCC relation which is an important health problem in our country. The HBV genome was purified from the Pt HBV 1,3 plazmid with terminally redundant DNA construct (HBV 1,3) and transferred to the one-celled embriyos of CD-1 mouse strain which were obtained after fertilization by pronuclear microenjection. The mice embriyos were transferred to the pseudopregnant CBGF 1 mouse strain that were under anesthesia. Transgenic offspring which were born after manipulation have been determined by polimerase chain reaction and dot-blot hybridization assays. HBV gene expression of transgenic mice has been shown to determine hepatitis B virus surface antigen by ELISA and immunohistochemical methods in their sera and livers and RT PCR was applied to the RNA samples which have been obtained from liver specimens. In this sudy, production of first transgenic animal in Turkey which is the transgenic mice containing and expressing HBV genome, has been accomplished.