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Raden Roro Sri Pudji Sinarni Dewi

Research Institute for Freshwater Fish Breeding and Aquaculture, Sukamandi

Author-ID : 3672307

Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology

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OPTIMAL ELECTROPORATION CONDITION FOR SPERM MEDIATED GENE TRANSFER IN STRIPPED CATFISH (Pangasionodon hypophthalmus) Raden Roro Sri Pudji Sinarni Dewi; Alimuddin Alimuddin; Agus Oman Sudrajat; Komar Sumantadinata; Sularto Sularto
Indonesian Aquaculture Journal Vol 5, No 1 (2010): (June 2010)
Publisher : Center for Fisheries Research, Agency for Marine and Fisheries Research and Human Resource

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.

IDENTIFICATION OF GROWTH HORMONE GENE OF Pangasionodon hypophthalmus Raden Roro Sri Pudji Sinarni Dewi; Agus Oman Sudrajat; Alimuddin Alimuddin; Komar Sumantadinata
Indonesian Aquaculture Journal Vol 4, No 1 (2009): (June 2009)
Publisher : Center for Fisheries Research, Agency for Marine and Fisheries Research and Human Resource

Identification of growth hormone (GH) gene in a target fish is the first step in the construction of “all fish genes transfer vector” to generate transgenic fish. The research was done to identify and characterize the GH gene of Pangasionodon hypophthalmus. There were several activities performed in identifying the GH gene: RNA extraction, cDNA synthesis, PCR amplification, and DNA fragment isolation. The characterizations were done using the nucleotide sequencing engine ABIPRISM 3100. The results were then analyzed using BLASTN/P and GENETYX version 7 program. The full-length GH gene of P. hypophthalmus was 1151 bp in length, coding for an open reading frame (ORF) of 603 bp. The 5’ and 3’ untranslated regions of the GH gene were 22 bp and 526 bp long, respectively. The GH gene of P. hypophthalmus had some common characteristics that are owned by GH genes, such as single tryptophan residue (W) on the 104th amino acid, 5 cysteine residues (C) on the amino acid 71, 135, 173, 190, and 198 and a motif of Asn-Xaa-Thr on C terminus which is the potential location for N-linked glycosilation. Polyadenylation signal (aataaa) was on the 14 bp at the upstream of polyadenylation location. Growth hormone of P. hypophthalmus consisted of over 200 amino acids from GH cDNA deduction. The highest proportion of amino acid composition was leusin (14%) while the lowest was tryptophan (0.5%).

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