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All Journal Indonesian Aquaculture Journal

Alimuddin Alimuddin

Department of Aquaculture, Faculty of Fisheries and Marine Science, Bogor Agricultural University, bogor

Author-ID : 3672308

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.

CLONING OF ProAV PROMOTER ISOLATED FROM TIGER PRAWN Penaeus monodon Andi Parenrengi; Alimuddin Alimuddin; Sukenda Sukenda; Komar Sumantadinata; Muhammad Yamin; Andi Tenriulo
Indonesian Aquaculture Journal Vol 4, No 1 (2009): (June 2009)
Publisher : Center for Fisheries Research, Agency for Marine and Fisheries Research and Human Resource

Promoter is a specific DNA sequence involved in the transcription of a particular gene. It is usually located in the upstream of the gene they regulate. Isolation and characterization of promoter is essentially needed in order to establish the sequence analysis and transcription factor that are used in the regulation of gene expression. The research was conducted to analyze the characteristics of Penaeus monodon anti viral gene promoter (ProAV) towards generation of auto-transgenic tiger prawn, P. monodon. ProAV promoter was isolated by PCR (Polymerase Chain Reaction) method and the purified DNA fragment was cloned into pGEM-T Easy cloning vector. The promoter sequence was characterized by using BLAST-N and Genetyx version 7 softwares. The results showed the success in isolating a promoter from tiger prawn of 368 bp in length. BLAST-N analysis showed that the sequence of isolated promoter has high similarity (95%-98%) compared to the other promoters in the GeneBank. The study revealed the existence of important transcription factors (TATA box, MRE, TCF-1, and other potential regulatory elements) are identified in the promoter sequence.

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%).

OVER-EXPRESSION OF GENE ENCODING FATTY ACID METABOLIC ENZYMES IN FISH Alimuddin Alimuddin; Goro Yoshizaki; Toshio Takeuchi; Odang Carman
Indonesian Aquaculture Journal Vol 3, No 2 (2008): (December 2008)
Publisher : Center for Fisheries Research, Agency for Marine and Fisheries Research and Human Resource

Eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have important nutritional benefits in humans. EPA and DHA are mainly derived from fish, but the decline in the stocks of major marine capture fishes could result in these fatty acids being consumed less. Farmed fish could serve as promising sources of EPA and DHA, but they need these fatty acids in their diets. Generation of fish strains that are capable of synthesizing enough amounts of EPA/DHA from the conversion of α-linolenic acid (LNA, 18:3n-3) rich oils can supply a new EPA/DHA source. This may be achieved by over-expression of genes encoding enzymes involved in HUFA biosynthesis. In aquaculture, the successful of this technique would open the possibility to reduce the enrichment of live food with fish oils for marine fish larvae, and to completely substitute fish oils with plant oils without reducing the quality of flesh in terms of EPA and DHA contents. Here, three genes, i.e. Δ6-desaturase-like (OmΔ6FAD), Δ5-desaturase-like (OmΔ5FAD) and elongase-like (MELO) encoding EPA/DHA metabolic enzymes derived from masu salmon (Oncorhynchus masou) were individually transferred into zebrafish (Danio rerio) as a model to increase its ability for synthesizing EPA and DHA. Fatty acid analysis showed that EPA content in whole body of the second transgenic fish generation over-expressing OmΔ6FAD gene was 1.4 fold and that of DHA was 2.1 fold higher (P<0.05) than those in non-transgenic fish. The EPA content in whole body of transgenic fish over-expressing OmΔ5FAD gene was 1.21-fold, and that of DHA was 1.24-fold higher (P<0.05) than those in nontransgenic fish. The same patterns were obtained in transgenic fish over-expressing MELO gene. EPA content was increased by 1.30-fold and DHA content by 1.33-fold higher (P<0.05) than those in non-transgenic fish. The results of studies demonstrated that fatty acid content of fish can be enhanced by over-expressing gene encoding enzymes involved in fatty acid biosynthesis, and perhaps this could be applied to tailor farmed fish as even better sources of valuable human food.

Co-Authors Agus Oman Sudrajat Andi Parenrengi Andi Tenriulo Goro Yoshizaki Komar Sumantadinata Muhammad Yamin Odang Carman Raden Roro Sri Pudji Sinarni Dewi Sukenda Sukenda Sularto Sularto Toshio Takeuchi

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