V. SIMPULAN DAN SARAN
5.2 Saran
Disarankan agar pada tahapan penelitian selanjutnya untuk memproduksi ikan mas transgenik F2 homosigot, dapat menggunakan ikan transgenik F1 dengan kategori memiliki peningkatan ukuran jauh lebih besar dibandingkan kontrol. Ikan transgenik homosigot berguna untuk produksi ikan transgenik secara massal.
DAFTAR PUSTAKA
Anzar M & Buhr MM. 2006. Spontaneous uptake of exogenous DNA by bull spermatozoa. Theriogenology 65: 683-690.
Arezzo F. 1989. Sea urchin sperm as vector for foreign genetic information. Cell Biol Int Rep 13: 391-404.
Arnesen AM, Toften H, Agustsson T, Stefansson SO, Handeland SO, and Björnsson BT. 2003. Osmoregulation, feed intake, growth and growth hormone levels in 0+ Atlantic salmon (Salmo salar L.) transferred to seawater at different stages of smolt development. Aquaculture 222: 167- 187.
Bardach JE, Ryther JH, and McLarney WO. 1972. Aquaculture: the farming and husbandry of freshwater and marine organisms. Wiley-Interscience, New York. 868 p.
Beardmore JA & Porter JS. 2003. Genetically modified organism and aquaculture. FAO Fisheries Circular. No. 989. Rome. 35p.
Björnsson BT, Ogasawara T, Hirano T, Bolton JP, and Bern HA. 1988. Elevated growth hormone levels in stunted Atlantic salmon, Salmo salar. Aquaculture 73: 275-281.
Björnsson BT, Stefansson GV, Berge ÅI, Hansen T, and Stefansson SO. 1998. Circulating growth hormone levels in Atlantic salmon smolts following seawater transfer: effects of photoperiod regime, salinity, duration of exposure and season. Aquaculture 168: 121-137.
Björnsson BT, Hemre GI, Bjørnevik M, and Hansen T. 2000. Photo period regulation of plasma growth hormone levels during induced smoltification of under yearling Atlantic salmon. Gen Comp Endocrinol 119: 17–25. Brackett BG, Baranaska W, Sawiicki W, and Koprosky. 1971. Uptake of
heterologous genome by mammalian spermatozoa and its transfer to ova through fertilization. Proc Natl Acad Sci 68: 353-357.
Buono RJ & Linser PJ. 1992. Transient expression of RSVCAT in transgenic zebrafish made by electroporation. Mar Mol Biol Biotechnol 1: 271-275. Calduch-Giner JA, Duval H, Chesnel F, Boeuf G, Perez-Sanchez J and Bouhard
D. 2000. Fish growth hormone receptor: Molecular characterization of two membrane-anchored forms. J Endocrine Society 142: 3269-3273.
Chen TT, Lu JK, and Kight K. 1995. Transgenic fish. In Meyers, RA (ed.). Molecular Biology and Biotechnology. VCH Publishers, Inc. 910-914.
Chen TT, Chen MJ, Chiou TT, and Lu JK. 2009. Transfer of foreign DNA into aquatic animals by electroporation. In Nakamura H (ed.). Electroporation and sonoporation in developmental biology. Springer. 229-237.
Chourrout D, Guyomard R, and Houdebine LM. 1986. High efficiency gene transfer in rainbow trout (Salmo gairdneri Rich) by microinjection into egg cytoplasm. Aquaculture 51: 143-150.
Ciereszko I, Johansson H, Hurry V, and Kleczkowski LA. 2001. Phosphate status affects the gene expression, protein content and enzymatic activity of UDP- glucose Pyrophosphorylase in wild type and pho mutants of Arabidopsis. Planta 212: 598-605
Collas P, Husebeye H and Alestrom P. 2000. Transferring foreign genes into Zebrafish eggs by microinjection. Transgenic Animal: Generation and Use. p119-122.
Cook JT, McNiven MA and Sutterlin AM. 2000. Metabolic rate of pre-smolt growth-enhanced transgenic Atlantic salmon Salmo salar. Aquaculture 188:33-45.
Devlin RH, Yesaki TY, Donaldson EM, Du SJ and Hew CL. 1994. Production of germline transgenic Pacific salmonids with dramatically increased growth performance. Canadian Journal of Fisheries Aquatic Sciences, 52 : 1376 – 1384.
Devlin RH, Yesaki TY, Donaldson EM, and Hew CL. 1995. Transmission and phenotypic effects of an antifreeze/GH gene construct in coho salmon (Oncorhynchus kisutch). Aquaculture 137: 161-169.
Dewi SPS, Rr. 2010. Studi over ekspresi gen penyandi hormon pertumbuhan melalui elektroporasi sperma untuk membuat ikan patin siam transgenik cepat tumbuh. [Tesis]. Bogor: Program Pascasarjana. Institut Pertanian Bogor. 74 hal.
Drennon K, Moriyama K, Kawauchi H, Small B, Silverstein J, Parhar I, and Shepherd B. 2003. Development of an enzyme-linked immuno sorbent assay for the measurement of plasma growth hormone (GH) levels in channel catfish (Ictalurus punctatus): assessment of environmental salinity and GH secretogogues on plasma GH levels. Gen Comp Endocrinol133: 314-322. Dunham RA. 2004. Aquaculture and Fisheries Biotechnology: Genetic
Approaches. CABI Publishing, Wallingford, hlm 66.
Du SJ, Gong Z, Fletcher GL, Shears MA, King MJ, Idler DR, and Hew CL. 1992. Growth enhancement in transgenic Atlantic salmon by the use of an” all fish” chimeric growth hormone gene construct. Biol Tech 10 : 176-180
Effendie MI. 1997. Biologi Perikanan. Yayasan Pustaka Nusantara. Yogyakarta.155 hal.
Einarsdottir IE, Sakata S, and Björnsson BT. 2002. Atlantic halibut growth hormone: structure and plasma levels of sexually mature males and females during photoperiod-regulated annual cycles. Gen Comp Endocrinol 127: 94- 104.
Faqih AR. 2011. Studi Rekayasa Genetik Melalui Elektroporasi DNA pada Sperma dalam Pembuatan Ikan Transgenik Air Tawar. [Desertasi]. Program Studi Ilmu-Ilmu Pertanian. Minat Bioteknologi. Program Pasca Sarjana. Universitas Brawijaya, Malang.
Farbridge KJ, Flett PA, and Leatherland JF. 1992. Temporal effect of restricted diet and compensatory increased dietary intake on thyroid function, plasma growth hormone levels and tissue lipid reserves of rainbow trout Onchorhynchus mykiss. Aquaculture 104: 157-174.
Fletcher GL & Davies PL. 1991. Transgenic fish for aquaculture. Genetic Engineering 13:331-371.
[FAO] Food and Agriculture Organization. 1999. The state of world fisheries and aquaculture 1998. Rome, Italy. 8 p.
[FAO] Food and Agriculture Organization. 2000. The state of world fisheries and aquaculture 2000. Rome, Italy. 10 p.
[FAO] Food and Agriculture Organization. 2010. Cultured Aquatic Species- Common carp. Food and Agriculture Organization, Roma.
Glick BR & Pasternak JJ. 2003. Molecular Biotechnology : Principles and Applications of Recombinant DNA. Third ed. ASM Press. Washington DC. Hackett PB. 1993. The Molecular Biology of Transgenic Fish. In: Hocachka and
Mommesen (Eds.). Biochemistry and Molecular Biology of Fishes 2 : 218- 221.
Hafez ESE. 1987. Reproduction in farm animals, 5th ed. Lea and Febiger. Philadelphia.
Hao F, Jia C, Jian L, Shao-Jun L, Yun L. 2006. Cloning of black carp β-actin and primarily detecting the function of its promoter region. Acta Genet Sinica 33(2): 133-140
Harvey BJ & Hoar BJ. 1979. The teory and practice of induce breeding in fish IDRC-TS 21e. International Development Research Centre. Ottawa, Canada. 48 p.
Inoue K, Yamashita S, Hata J, Kabeno S, Asada S, Nagahisa E, Fujita T. 1990. Electroporation as a new technoloque for producing transgenic fish. Cell Differ Dev 29: 123-128.
Jamieson BGM. 1990. Fish evolution and systemic : Evidence from spermatozoa (with a survey of Lophophorate, Echinoderm and Prrotochordate sperm) and an account of gamete cryopreservation. Cambridge University Press. New York.
Jourdan S, Fontaine P, Boujard T, Vandeloise E, Gardeur JN, Anthouard M, Kestemont P. 2000. Influence of day length on growth, heterogenity, gonad development, sexual steroid, and thyroid levels, and N and P budgets in Perca fluviatilis. Aquaculture 186: 253-265
[KKP] Kementerian Kelautan dan Perikanan. 2010. Statistik Menakar 353. Direktorat Jenderal Perikanan Budidaya. Jakarta.
Kinoshita M & Ozato K. 1995. Cytoplasmic microinjection of DNA into fertilized Medaka Oryzias latipeseggs. The Fish Biology Journal MEDAKA 7:59-64. Knight DE. 1981. Rending cells permeable to exposure to electric fields. Tech
Cell Physiol 113: 1-10.
Knight DE & Scrutton MC. 1986. Gaining access to the cytosol: the technique and some application of electropermeabilization. Biochem J 234: 497-506. Kato K, Takagi M, Tamaru Y, Akiyama S, Konishi T, Murata O, and Kumai H.
2007. Construction of an expression vector containing a β-actin promoter region for gene transfer by microinjection in Red Sea Bream Pagrus major. Fisheries Science 73: 440-445.
Kobayashi SI, Alimuddin, Morita T, Miwa M, Lu J, Endo M, Takeuchi T and Yoshizaki G. 2007. Transgenic nile tilapia Oreochromis niloticus over- expressing growth hormone show reduced ammonia excretion. Aquaculture 270: 427-435.
Koolman J & Rohm KH. 2001. Atlas berwarna dan teks biokimia. Wanandi, penerjemah. Jakarta: Hipokrates. Terjemahan dari: Color Atlas of Biochemistry.
Lanes CFC, Sampaio LA, and Marins LF. 2009. Evaluation of Dnase activity in seminal plasma and uptake of exogenous DNA by spermatozoa of Brazilian flounder (Paralichthys orbignyanus). Theriogenology 71: 525-533.
Lavitrano M, Camaioni A, Faizo V, Dolci S, Farace MG, and Spadafora C. 1989. Sperm cells as vectors of foreign DNA: genetic transformation of mice. Cell 57: 717-723.
Lavitrano M, Marcho B, Maria GC, Roberto G, Stefano M, Alessia. 2006. Sperm mediated gene transfer, Reproduction, fertility and development. CSIRO Publising 18: 19-23
Le Gac F, Blaise O, Fostier A, Le Bail P, Loir M, Mourot B and Weil C. 1993. Growth hormone (GH) and reproduction: a review. Fish Physiol Biochem 11: 219-232.
Lin S, Gaiano N, Culp P, Burns JC, Friedman T, and Yee JK. 1994. Integration and germ-line transmission of pseudo typed retroviral vector in zebra fish. Science 265: 666-669.
Lu JK, Fu BH, Wu JL, Chen TT. 200 2. Production of transgenic silver sea bream (Sparus sarba) by different gene transfer methods. Mar Biotechnology 4: 328-337.
MacLean N & Donaldson EM. 1993. The role of somatotropin in growth in poikilotherms. In Martin P, Scanes CG, Pang PKT (eds). The endocrinology of growth, development and metabolism in vertebrates. Academic Press. Matty AJ. 1985. Fish endocrinology. Croom Helm London and Sydney Timber
Press. Portland, Oregon. 267 p.
Nam YK, Noh JK, Cho YS, Cho HJ, Cho KN, Kim CG and Kim DS. 2001. Dramatically accelerated growth and extraordinary gigantism of transgenic mud loach Misgurnus mizolepis. Transgenic Research 10:353-362.
Nordgarden U, Hansen T, Hemre GI, Sundby A, Björnsson BT. 2005. Endocrine growth regulation of adult Atlantic salmon in seawater: the effects of light regime on plasma growth hormone, insulin-like growth factor-I, and insulin levels. Aquaculture 250: 862-871.
Ohta Y, Kidd MT, and T Ishibashi. 2001. Embryo growth and amino acid concentration profiles of broiler breeder eggs, embryos, and chicks after in ovo administration of amino acids. Poultry Science 80: 1430-1436
Ozato K, Kondoh H, Inohara H, Iwamatsu T, Wakamatsu Y, and Okada TS. 1986. Production of transgenic fish: introduction and expression of chicken δ- crystallin gene in medaka embryos. Cell Differ 19: 237-244.
Palmitter RD, Brinster RL, Hammer RE, Trumbauer ME, and Rosenfeld MG. 1982. Dramatic growth of mice that developed from eggs microinjected with metallothionein-growth hormone fusion genes. Nature 30: 611-615.
Peter RE & Marchant TA. 1995. The Endocrinology of growth in carp and related spesies. Aquaculture 129: 299-321.
Powers DA, Herefort L, Cole T, Chen TT, Lin CM, Knight K, Creech K, and Dunham R. 1992. Electroporation method for transferring genes into the gametes of zebrafish (Brachydanio rerio), channel catfish (Ictalurus punctatus) and common carp (Cyprinus carpio). Mol Mar Biol Biotechnol 1:301-308.
Price SA & Wilson LM. 1995. Patofisiologi, Konsep klinis proses-proses penyakit, edisi 4 buku II. Penerbit Buku Kedokteran EGC. 65hal.
Rahman MA, Hwang G, Razak SA, Sohm F, and MacLean N. 2000. Copy number dependent transgene expression in hemizygous and homozygous transgenic tilapia (Oreochromis niloticus). Transgenic Research 9: 417-427. Rand-Weaver M & Kawauchi H. 1993. Growth hormone, prolactin and
somatolactin: a structural overview. In Hochachka PW, Mommsen TM (eds). Biochemistry and Molecular Biology of Fishes 2: 39-56.
Robert S, Barry T, Malison J, and Goetz F. 2004. Production of recombinantly Derived growth hormone antibody and the characterization of growth hormone levels in yellow perch. Aquaculture 232: 591-602.
Sakamoto T & Hirano T. 1993. Expression of insulin-like growth factor I gene in osmoregulatory organs during seawater adaptation of the salmonid fish: possible mode of osmoregulatory action of growth hormone. Proc Natl Acad Sci USA 90: 1912–1916.
Sarmasik A, Jang IK, Chung CZ, Lu JK, and Chen TT. 2001. Transgenic live- bearing fish and crustacean produced by transforming immature gonads with replication-defective pantropic retrovial victor. Mar Biotechnol 3: 470-477. Sarmasik A. 2003. Application of gene transfer technology for genetic
improvement of fish. Turk J Zool 27: 1-6.
Sin FYT, Mukherjee UK, McKenzie JC and Sin IL. 1995. Abalone sperm-DNA interaction. In: Proceedings of International Symposium on Biotechnology Applications in Aquaculture. National Taiwan University. 95-99.
Sin FYT, Walker SP, Symonds JE, Mukherjee UK, Khoo JGI, and Sin IL. 2000. Electroporation of salmon sperm for gene transfer: efficiency, reliability, and fate of transgene. Mol Repro and Dev 56: 285-288.
Spadafora C. 1998. Sperm cells and foreign DNA: a controversial relation. Bioessays 20: 955-964.
Stoss J & Donaldson EM. 1982. Preservation of Fish Gametes. In : CJJ Richter and HJ Th. Goos, (Comp.), “Proceedings of the International Symposium on Reproductive Physiology of Fish, Wageningen, the Netherlands, 2-6 August, 1982” Pudoc, Wageningen, 114-122p
Subyakto S. 2010. Efektivitas dan Efisiensi Transfeksi, Mikroinjeksi dan Elektroporasi untuk Transfer Gen Hormon Pertumbuhan pada Ikan Kerapu Tikus (Cromileptes altivelis). [Disertasi]. Program Studi Ilmu-Ilmu Pertanian. Minat Lingkungan Pesisir Dan Kelautan. Program Pasca Sarjana. Universitas Brawijaya, Malang. 110hal
Swift DR & Pickford GE. 1965. Seasonal variations in the hormone content of the pituitary gland of the perch (Perca fluviatilis L.). Gen Comp Endocrinol 5: 354-365.
Symonds JE, Walker SP, Sin FYT. 1994. Development of mass gene transfer method in chinook salmon: optimization of gene transfer by electroporated sperm. Mol Mar Biotechnol 3: 104-111.
Tabarés G, Jung K, Reiche J, Stephan C, Lein M, Peracaula R, de Lorens R, and Hoesel W. 2007. Free PSA forms in prostatic tissue and sera of prostate cancer patients: Analysis by 2-DE and western blotting of immunopurified samples. Clin Biochem 40:343-350
Takahashi A, Ogasawara T, Kawauchi H, Hirano T. 1991. Effects of stress and fasting on plasma growth hormone levels in the immature rainbow trout. Nippon Suisan Gakkaishi 57: 231-235.
Tatsuya S & Hirano T. 1993. Expression of insulin-like growth factor I gene in osmoregulatory organs during seawater adaptation of the salmonid fish: possible mode of osmoregulatory action of growth hormone. Proc Natl Sci USA 90: 1912-1916.
Tave D. 1986. Genetic for Fish Hatchery Managers. Second Edition. An AVI Book. Published by Van Nostrand Reinhold, New York. 415 pp.
Toelihere MR. 1981. Fisiologi Reproduksi pada Ternak. CV Angkasa. Bandung. 109 hal.
Tsai HJ, Tseng FS, and Liao IC. 1995a. Electroporation of sperm to introduce foreign DNA into the genome of loach (Misgurnus anguilicaudatus). Can J Fish Aquat Sci 52: 776-787.
Tsai HJ, Lin KL, Kuo JC, Chen SW. 1995b. Highly efficient expression of fish growth hormone by Escherichia coli cells. Appl Environ Microbiol 61:4116-4119.
Tsai HJ. 2000. Electroporated sperm mediation of gene tranfer system for finfish and shellfish. Mol Repro Dev 56: 281-284.
Tsong TY. 1983. Voltage modulation of membrane permeability and energy utilization in cells. Biosci Rep 3: 487-505.
Van der Kraak G, Rosenblumm PM, and Peter RE. 1990. Growth hormone- dependent potentiation of gonadotropin stimulated steroid production by ovarian follicles of the goldfish. Gen Comp Endocrinol 79: 233-239.
Walsh G. 2002. Proteins: Biochemistry and biotechnology. John Wiley & Sons, LTD.
Wu G, Sun Y, and Zhu Z. 2003. Growth hormone gene transfer in common carp. Aguat Living Resour 16: 416-420.
Yaskowiak ES, Shears MA, Agarwal-Mawal A, Fletcher GL. 2006. Characterization and multi-generational stability of the growth hormone transgene (EO-1α) responsible for enhanced growth rates in Atlantic salmon. Transgenic Research 15: 465-480
Yazawa R, Hirono I, Yamamoto E, and Aoki T. 2005. Gene transfer for Japanese flounder fertilized eggs by particle gun bombardment. Fisheries Sci 71: 869- 874.
Zaneveld LJD. 1978. The Biology of Human Spermatozoa, In Wynn E.D. Obsterric and Gynecology Annual. Appleton Century Croft. Chicago. p15- 40.
Zhu Z, Li G, He L, Chen S. 1985. Novel gene transfer into fertilized eggs of goldfish Carassius auratus L.1758. J Appl Ichthyol 1: 31-34
Lampiran 1. Abstrak makalah presentasi oral dalam Forum Inovasi Teknologi Akuakultur, tanggal 19-21 Juli 2011 di Bali.
PERTUMBUHAN IKAN MAS TRANSGENIK KETURUNAN PERTAMA
Nurly Faridah1,2, Alimuddin3, Dian Hardiantho2, Arief Eko Prasetiyo, Dwi Hani Yanti2, Irvan Faizal4, dan Komar Sumantadinata3
1. Mahasiswa Program Magister, Program Studi Akuakultur, Departemen Budidaya Perairan, FPIK, IPB Bogor.
2. Balai Besar Pengembangan Budidaya Air Tawar, Sukabumi. 3. Departemen Budidaya Perairan, FPIK, IPB Bogor.
4. Badan Pengkajian dan Penerapan Teknologi, Serpong, Tangerang.
ABSTRAK
Penelitian ini dilakukan untuk memproduksi ikan mas transgenik keturunan pertama (F1) dan menganalisis ekspresi gen hormon pertumbuhan ikan nila (nHP) yang ditransfer. Gen nHP yang ekspresinya dikendalikan oleh promoter -aktin ikan medaka (mBA) ditransfer ke sperma ikan mas menggunakan metode elektroporasi. Sperma tersebut selanjutnya digunakan untuk membuahi telur ikan mas untuk menghasilkan ikan transgenik founder (F0). Dengan menggunakan metode PCR dengan primer spesifik untuk gen nHP, diperoleh 22 ekor ikan mas jantan transgenik F0 dan 85 ekor ikan mas betina yang membawa gen nHP di siripnya. Selanjutnya, ikan mas jantan transgenik F0 dikawinkan dengan ikan non-transgenik untuk memproduksi F1. Dari hasil identifikasi menggunakan metode PCR dan pengukuran bobot tubuh ikan, diperoleh 5 ekor dari 84 ekor ikan transgenik F1 yang memiliki bobot sekitar 100 - 177% lebih tinggi daripada ikan non-transgenik. Analisis RT-PCR menunjukkan bahwa ikan transgenik F1 tersebut memiliki ekspresi gen nHP yang tinggi pada sirip ekornya, sementara pada ikan non-transgenik tidak ada ekspresi. Dengan demikian, peningkatan pertumbuhan bobot ikan mas transgenik tersebut terkait dengan ekspresi gen nHP, dan budidaya ikan mas transgenik nHP ini sangat berpotensi untuk mendukung pencapaian target produksi ikan mas nasional 2014.
Lampiran 2. Prosedur pelaksanaan elektroporasi.
GambarGene Pulser Xcell Unitdengan komponen PC Module (A), Pulse button (B),LCD Monitor (C), ShockPod (D), Key Pad (E), dan kabel power (F)
1. Menghubungkan Gene Pulser Xcell Unit dengan CE Module, PC Module, dan ShockPod. Sebelum menyambungkan bagian-bagian tersebut, pastikan Gene PulserXcell Unit dalam kondisi OFF.
2. Menghubungkan kabel power dengan sumber listrik AC, kemudian menyalakan unit
3. Memasukkan sampel yang akan dianalisis ke dalam cuvvet dan memasukkan ke dalam ShockPod, kemudian menutup dan mengunci dengan sempurna karena pulse tidak akan terkirim jika penutup ShockPod dalam kondisi terbuka.
4. Pada menu HOME (tekan tombol HOME untuk masuk ke menu HOME) 5. Menekan ENTER untuk memilih eksponensial decay
6. Menekan nomor 2, kemudian ENTER untuk memilih eksponensial decay tetapi spesifikasikan konstanta waktunya terlebih dahulu
7. Menekan nomor 3, kemudian ENTER untuk memilih square wave.
8. Gunakan tombol panah naik dan turun untuk bergeser dan memilih nilai parameter pada layar. Saat parameter terpilih, gunakan keypad untuk memasukkan nilai yang diinginkan, tekan ENTER untuk setuju.
A B E D F C
9. Ketikan nilai parameter yang diinginkan selesai dimasukkan semua, tombol PULSE pada Gene PulserXcell menjadi aktif.
10. Menekan tombol PULSE untuk mengelektroporasi sampel.
11. Setelah penghantaran pulse, layar LCD menampilkan hasil pada tampilan Protocol Result.
12. Menekan tombol BACK untuk kembali ke tampilan Protocol Detail dan beralih ke pulse yang lain.
13. Hasil 100 pulse terakhir termasuk parameter pulse tersimpan dalam memori Gene Pulser Xcell dan dapat diakses dari program Data Management
14. Untuk menyimpan protokol yang telah dibuat, saat tampilan Protocol Detail terbuka, tekan SAVE. Selanjutnya ikuti petunjuk yang muncul di layar untuk menentukan lokasi penyimpanannya.
15. Mematikan unit dengan menekan tombol POWER setelah selesai digunakan, cabut kabel dari sumber listrik (AC).
PROTOCOL DETAIL: EXPONENTIAL Voltage (V)
XXXX
Capacitance (uF) XXXX Resistance (ohm) XXXX Cuvvette (mm) X Exponential Decay Protocol Detail Screen
Lampiran 3. Sperma ikan mas setelah perlakuan elektroporasi dengan DNA pmβa-tiGH, diamati di bawah mikroskop pada pembesaran 40x10 (A) dan 100x10 (B). Sperma mati ditunjukkan dengan panah , sedangkan sperma hidup ditunjukkan dengan panah .
Lampiran 4. Perkembangan embrio ikan mas hasil fertilisasi telur dengan sperma yang sudah dielektroporasi
(A) (B) (C)
(D) (E) (F)
(G) (H)
Keterangan: A = embrio,
B = embrio, fase 2 sel C = embrio, fase 4 sel
D = embrio, fase gastrula, 2 jam setelahpembuahan E & F = emrio, fase organogenesis
Lampiran 5. Data hasil elektroporasi pada program 1 dan program 2, serta efektivitasnya terhadap motilitas dan viabilitas sperma, derajat pembuahan (FR) dan derajat penetasan (HR) telur ikan mas.
P ro g ra m 1 Ulangan [DNA] ng/μl Vol. DNA Vol. Sperma Lar Fis Volt setting Volt actual Droop (%) Motilitas Sperma Viabilitas Sperma DNA pada Sperma* ∑ Telur (butir) FR (%) ∑ Telur Terbuahi (butir) HR (%) ∑ Telur Menetas (butir) 1 25 10 50 530 50 V 49 2 95 99 ada 1000 91,7 917 88,2 809 2 25 10 50 530 50 V 49 2 90 96 ada 1000 89,3 893 85,3 762 3 25 10 50 530 50 V 48 4 90 96 ada 1000 89,9 899 84,7 826 Kontrol 0 (10) TE 50 530 50 V 49 2 90 96 - 1000 93,4 934 88,4 761 P ro g ra m 2 Ulangan [DNA] ng/μl DNA Vol. Vol. Sperma Lar Fis Volt setting Volt actual Droop (%) Motilitas Sperma Viabilitas Sperma DNA pada Sperma* ∑ Telur (butir) FR (%) ∑ Telur Terbuahi (butir) HR (%) ∑ Telur Menetas (butir) 1 50 10 50 530 40 V 38 5 95 98 ada 1000 91,4 914 85,7 783 2 50 10 50 530 40 V 36 10 90 96 ada 1000 86,2 862 83,7 721 3 50 10 50 530 40 V 38 5 95 99 ada 1000 88,7 887 84,3 748 Kontrol 0 (10) TE 50 530 40 V 36 10 90 98 - 1000 93,7 937 86,6 811
Lampiran 6. Kelangsungan hidup (SR) larva (5 hari) dan benih (2 bulan) ikan mas dari telur yang dibuahi dengan sperma yang dielektroporasi menggunakan program 1 dan program 2
Progra m 1 Ulangan SR 5 hari (%) ∑ larva 5 hr (ekor) SR 2 bulan (%) ∑ benih 2 bln (ekor) 1 67,2 544 38,3 208 2 66,6 507 40,2 204 3 72,8 601 36,6 220 Kontrol 68,3 520 38,6 201 Progra m 2
Ulangan SR 5 hari (%) ∑ larva 5 hr (ekor) SR 2 bulan (%) ∑ benih 2 bln (ekor)
1 67,7 530 35,8 190
2 65,3 471 39,3 185
3 69,1 517 37,3 193
Lampiran 7. Data hasil analisis DNA tiGH pada sirip individu F1 berumur 2 bulan dan persentase ikan transgenik F1.
Pr ogr am 1 Ulangan ∑ sampel dianalisis (ekor)
Hasil analisis (ekor) Persentase ikan transgenik F1
(%)
∑ sampel
positif ∑ sampel negatif
1 208 99 109 47,60 2 204 106 98 51,96 3 220 118 102 53,64 Rataan 210,67 107,67 103,00 51,06 Pr ogr am 2 Ulangan ∑ sampel dianalisis (ekor)
Hasil analisis (ekor) Persentase ikan transgenik F1 (%) ∑ sampel positif ∑ sampel negatif 1 190 58 132 30,53 2 185 60 125 32,43 3 193 56 137 29,02 Rataan 189,33 58,00 131,33 30,66
Lampiran 8. Hasil analisis DNA pada gonad calon induk F0 Progra m 1 Ulangan ∑ benih calon F0 (ekor) SR 8 bulan (%) ∑ F0 dianalisis (ekor)
Hasil analisis (ekor) germline
transmitte r (%) ∑ sampel positif ∑ sampel negatif 1 99 70,50 70 36 63 51,58 2 106 64,90 69 28 78 40,70 3 118 66,20 78 43 75 55,05 Rerata 108 67,20 72 35,67 72,00 49,11 Total 323 - 217 107 216 49,38 Progra m 2 Ulangan ∑ benih calon F0 (ekor) SR 8 bulan (%) ∑ F0 dianalisis (ekor)
Hasil analisa (ekor)
germline transmitte r (%) ∑ sampel positif ∑ sampel negatif 1 190 69,3 132 44 88 33,42 2 185 62,7 116 30 86 25,86 3 193 71,3 138 51 87 37,06 Rerata 189 67,77 128 41,67 86,76 32,11 Total 568 - 385 125 260 32,44
Lampiran 9. Hasil identifikasi tiGH dan laju transmisi pada keturunan pertama (F1) dari tiap induk (F0).
Induk Jantan Nomor Tagging Kondisi gonad Jumlah F1 dianalisis (ekor) jumlah F1 positif sirip (ekor) Laju transmisi pada F1 (%) 1 4612 706 431 Matang gonad 92 48 52,174
2 460F 205 92A Matang gonad 60 40 66,667
3 460D 6C6 F34 Matang gonad 89 42 47,191
4 4612 237 605 Matang gonad 28 22 78,571
5 460F 066 B1F Matang gonad 14 7 50,000
Lampiran 10. Data pertumbuhan individu keturunan pertama (F1) selama 60 hari pemeliharaan
induk tiGH Bobot rataan individu hari ke- (g) α
0 20 40 60 1 Transgenik 5,25 12,33 26,93 50,12 3,80% Non-transgenik 5,01 9,68 19,33 33,21 3,20% 2 Transgenik 2,86 7,38 14,34 25,58 3,72% Non-transgenik 2,74 5,92 11,17 18,92 3,27% 3 Transgenik 2,95 7,62 15,56 30,38 3,96% Non-transgenik 2,98 5,64 11,72 21,26 3,33% 4 Transgenik 4,73 12,33 26,71 45,94 3,86% Non-transgenik 2,62 5,60 12,67 24,67 3,81% 5 Transgenik 18,00 47,33 92,46 160,84 3,72% Non-transgenik 10,07 22,33 46,50 87,33 3,67%
1 Lampiran 11. Tingkat kelangsungan hidup individu keturunan pertama (F1) dari
tiap induk founder (F0) selama 60 hari pemeliharaan
induk tiGH Kelangsungan hidup pada hari ke- (%)
0 20 40 60 1 Transgenik 100,0 100,0 100,0 100,0 Non-transgenik 100,0 100,0 100,0 100,0 2 Transgenik 100,0 100,0 96,7 89,7 Non-transgenik 100,0 93,1 89,7 82,8 3 Transgenik 100,0 100,0 100,0 90,0 Non-transgenik 100,0 100,0 96,7 86,7 4 Transgenik 100,0 100,0 100,0 100,0 Non-transgenik 100,0 90,9 90,9 72,7 5 Transgenik 100,0 100,0 100,0 100,0 Non-transgenik 100,0 100,0 100,0 100,0 58