| Autor: |
Rajpurohit SK; Georgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA., Gopal A; Department of Medicine, Division of Cardiology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA., Mon MY; Georgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA., Patel NG; Department of Pathology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA., Arora V; Department of Medicine, Division of Cardiology, Medical College of Georgia, Augusta University, Augusta, GA 30912, USA. |
| Abstrakt: |
The zebrafish provided an excellent platform to study the genetic and molecular approach of cellular phenotype-based cardiac research. We designed a novel protocol to develop the transparent transgenic zebrafish model to study annexin-5 activity in the cardiovascular function by generating homozygous transparent skin Casper( roy -/- ,nacre -/- ) ; myl7:RFP; annexin-5:YFP transgenic zebrafish. The skin pigmentation background of any vertebrate model organism is a major obstruction for in vivo confocal imaging to study the transgenic cellular phenotype-based study. By developing Casper( roy -/- ,nacre -/- ) ; myl7; annexin-5 transparent transgenic zebrafish strain, we established time-lapse in vivo confocal microscopy to study cellular phenotype/pathologies of cardiomyocytes over time to quantify changes in cardiomyocyte morphology and function over time, comparing control and cardiac injury and cardio-oncology. Casper contributes to the study by integrating a transparent characteristic in adult zebrafish that allows for simpler transparent visualization and observation. The Casper( roy -/- ,nacre -/- ) transgenic progenies developed through cross-breeding with the transgenic strain of Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP) . Confocal and fluorescent microscopy were being used to obtain accurate, precise imaging and to determine fluorescent protein being activated. This study protocol was conducted under two sections; 1.1: Generation of homozygous Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP) ; Casper( roy -/- ,nacre -/- ) zebrafish (generation F01-F06) and 1.2: Screening and sorting the transparent transgenic progeny and in vivo imaging to validate cardiac morphology through in vivo confocal imaging. We coined the newly developed strain as Tg(UAS:SEC-Hsa.ANXA5-YFP,myl7:RFP) ; Casper( roy -/- ,nacre -/- ) gmc1 . Thus, the newly developed strain maintains transparency of the skin throughout the entire life of zebrafish and is capable of application of a non-invasive in vivo imaging process. These novel results provide an in vivo whole organism-based platform to design high-throughput screening and establish a new horizon for drug discovery in cardiac cell death and cardio-oncology therapeutics and treatment. |
