Big size betta (Giant) have a high economic value compared to normal size betta, and over expression of growth hormone gene can produce giant fish. As an initial step of giant transgenic betta productions, this study was conducted in order to obtain DNA plasmid concentration which provide higher hatching and survival rate of betta larvae. Construction of PhGH pCcBA gene contains growth hormone gene of Siamese catfish (PhGH) and it is controlled by the CCBA promoter. Betta imbellis broodstocks were spawned naturally, and embryos were collected 1-2 minutes after spawning time. One hundred embryos were dipped in 2 mL of transfectan X-treme gene which containp CcBA-PhGH construction genes (50 µg/mL), on room temperature for about 30 minutes. Treatments on this study were different transfectant : DNA plasmid ratiosnamely:A (0,75 µL: 0,25 µL); B (0,75 µL : 0,50 µL); C (0,75 µL: 0,75 µL), D as Control 1(without transfectant, 0,25 µL DNA); E.as Control 2(0,75 µL transfectant, without DNA), and Fas control 3 (without transfectant and without DNA). Every treatments was repeated three times. Transfection embryos were hatched on a container (1L Volume). Study results showed that hatching rate and larvae survival rate (4 days after hatching) on treatment A were the same with the control, but slightly higher than B and C treatments. PCR analysis with DNA template showing that PhGH gene were found on embryos and larvae (pooled sample) of treatment A, B and C. Furthermore, RT-PCR analysis showing the existence of mRNA PhGH expression on embryos and larvae (pooled sample). Therefore, embryo transfection with transfectant ratio 0,75 µL and DNA 0,25 µLshowing the best results.
ABSTRAKPenelitian dilakukan untuk mengidentifikasi keberhasilan introduksi gen penyandi hormon pertumbuhan (Growth Hormone, GH) pada induk F-0 ikan Betta imbellis. Ikan transgenik F-0 dibuat dengan menggunakan metode transfeksi. Identifikasi dilakukan menggunakan metode RT-PCR. RNA total diekstraksi dari embrio pooled sample hasil persilangan induk transgenik dan non-transgenik. Berdasarkan analisis ekspresi gen pada embrio juga menunjukkan adanya aktivitas ekspresi gen GH pada semua perlakuan dibandingkan dengan kontrol (embrio hasil persilangan non-transgenik x non-transgenik). Jumlah individu induk F-0 yang membawa gen GH eksogen berdasarkan analisis PCR dengan DNA template dari sirip ekor adalah sebanyak 16%. Individu positif membawa gen GH eksogen tersebut dibesarkan lebih lanjut untuk memproduksi Betta imbellis transgenik F-1. Kandidat ikan transgenik jantan F-0 dikawinkan dengan ikan non-transgenik betina, sedangkan transgenik F-0 betina dikawinkan dengan non-transgenik jantan. Sebanyak 30-50 butir embrio hasil pemijahan F-0 digabung, kemudian DNA genom diekstrak. Sebagian embrio digunakan untuk ekstraksi RNA total untuk analisis ekspresi mRNA GH eksogen. Hasil analisis PCR menunjukkan bahwa semua sampel embrio dari induk transgenik F-0 dapat terdeteksi gen GH eksogen, sedangkan untuk kontrol (non-transgenik) tidak terdeteksi. Ekspresi mRNA juga terdeteksi pada embrio F-1. Dengan demikian, metode transfeksi embrio Betta imbellis efektif digunakan untuk menghasilkan ikan transgenik, dan sangat berpotensi menghasilkan individu F-1 Betta imbellis dengan pertumbuhan lebih cepat.KATA KUNCI: Betta imbellis; hormon pertumbuhan; identifikasi induk F-0 ABSTRACT:Identification of transgenic founder betta fish (Betta imbelis) carried growth hormone gene. By: Eni Kusrini, Alimuddin, Muhammad Zairin Jr., and Dinar Tri Sulistyowati The study was conducted to identify the successful introduction of the growth hormone gene (Growth Hormone, GH) on the F-0 Betta imbellis broodstock. The F-0 transgenic fish was made through transfection methods. Identification was done using RT-PCR method. Total RNA was extracted from pooled embryos sample. Based on the analysis of gene expression in embryos also showed activity GH gene expression in all treatments compared to the control (nontransgenic x non-transgenic). The number of individuals F-0 which carried exogenous GH gene by PCR analysis of the
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