COMPARISON OF THREE DIFFERENT TECHNIQUES OF GENE TRANSFER IN HUMPBACK GROUPER ( ) CROMILEPTES ALTIVELIS

Abstract

Humpback grouper is one of the most cultured fishes in Asia, including Indonesia. The main problemfaced by humpback culture is its slow growth rate.One of themethods that will be more effective and efficient to solve the problem is using transgenic technique. This study was conducted to determine the effectiveness of transfection,microinjection and electroporation techniques on gene transfer in humpback grouper. transfection was performed by incubating sperm to the foreign DNA (pktBP-ktGH gene construct)-transfectant complex solution, while was by injecting those complex solution into testis of mature males. Microinjection was conducted in 2-4 cell stage embryos using 25 μg/ml of foreign DNA solution, and duration of injection was 1, 2 and 3 seconds. Electroporation by 50 V, 30 ms of pulse length, 5 of pulse number and 0.1 of pulse interval was performed to sperm using three DNA concentration of 5, 10 and 20 μg/ml. The incorporation of foreign DNA in sperm and embryos were analyzed using PCR method. Based on PCR analysis, an optimum DNA concentration for electroporation was 10 μg/ml. Limited number of embryos could be microinjected during 20-30 min to reach 2-4 cell stage. Microinjection for 1 second showed higher survival rate of embryos, although none or very low number of larvae was hatched. Transfast was an effective DNA delivery reagent for humpback grouper sperm. Foreign DNA could be detected in sperm from two out of ten transfected fish at least 36 hours post transfection (hpt). By transfection, foreign DNA was detected in sperm at 48 hpt 25 C incubation temperature. Our study revealed that transfection, microinjection as well as electroporation could be used as transgenesis methods in humpback grouper. By means of simplicity and efficacy, however, electroporationwas an appropriate gene transfermethod. o In vitro in vivo in vivo in vitro