OPTIMAL ELECTROPORATION CONDITION FOR SPERM MEDIATED GENE TRANSFER IN STRIPPED CATFISH (Pangasionodon hypophthalmus)
Abstract: The success of transgenic
fish production has been achieved through eggs fertilization using
electroporated sperms carrying exogenous DNA. This study was conducted in order
to obtain the optimal electroporation condition for stripped catfish sperm. A
plasmid containing green fluorescent protein (GFP) gene driven by carp β-actin
promoter was transferred into sperm using electrophoresis method towards
transgenic stripped catfish (Pangasionodon hypophthalmus) production.
Electroporation was carried out using square wave shock with pulse length of 30
ms and pulse interval of 0.1 sec. Treatments are combination between voltage
(50 V, 75 V, and 100 V) and pulse number (1 and 3). Exogenous DNA concentration
used was 10 μg/mL of Tris-EDTA. Results showed that increasing the voltage from
50 to 100 decreased sperm motility, while pulse number did not affect sperm
motility. Voltage of 50 gave the best motility of sperm, although sperm
viability relatively similar between treatments and control except at 100 V
with 3 pulses number. Further, electroporation-treated sperms were able to
fertilize eggs. Higher hatching rate of eggs was obtained in electroporation
treatment at 50 V with pulse number of 1 and 3. The persistence of transferred
GFP was detected in electroporated and incubated sperms (control). However, GFP
was only detected in larvae from eggs that were fertilized by electroporated
sperm. Thus, electroporation could be applied to produce transgenic stripped
catfish.
Keywords: electroporation;
sperm; Pangasionodon hypophthalmus
Author: Raden Roro Sri Pudji
Sinarni Dewi, Alimuddin Alimuddin, Agus Oman Sudrajat, Komar Sumantadinata,
Sularto
Journal Code: jpperikanangg100009