Efektivitas promoter keratin lebih baik dibandingkan dengan promoter heatshock pada ikan nila Oreochromis niloticus. Promoter keratin memiliki pola aktivitas sementara relatif sama dengan promoter heatshock, namun pada promoter keratin awal aktivitasnya terjadi lebih cepat.
5.2 Saran
Untuk pembuatan ikan nila transgenik secara umum, sebaiknya digunakan promoter keratin, karena promoter ini memiliki efektifitas yang lebih baik.
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Lampiran 1. Embriogenesis ikan nila O.niloticus (Fujimura & Okada, 2007)
Lampiran 2. Pemijahan ikan nila dengan sistem semi buatan
1
2
3
4
1. Identifikasi induk jantan dan betina yang siap memijah (ditandai dengan adanya tonjolan pada daerah lubang urogenitalnya
2. Kedua induk kemudian dicampurkan dengan cara memindahkan induk jantan pada akuarium induk betina
3. Setelah induk terjadi pemijahan secara alami, kemudian dilakukan stripping pada induk betina dan induk jantan
4. Telur yang telah dibuahi, kemudian didiamkan beberapa saat guna pembuahan berlangsung dengan baik
Lampiran 3. Persiapan alat mikroinjeksi
Keterangan :
A. 1 set alat mikroinjeksi B. Injektor
C. Gel plat mikroinjeksi dan telur yang telah disusun guna diinjeksi D. Micromanipulator
A B
D C
Lampiran 4. Konstruksi DNA (Keratin-GFP) dan (Heatshock-GFP)
a. Konstruksi DNA Keratin-GFP (Yazawa et al.,2005)
b. Konstruksi DNA Heatshock-GFP (Yamamoto dan Yoshizaki, belum dipublikasikan)
Lampiran 5. Persiapan kontruksi DNA berupa perbanyakan dan isolasi plasmid
Lampiran 6. Proses memasukkan DNA kedalam jarum mikroinjeksi (Loading DNA)
1. Larutan DNA diambil dengan menggunakan mikrotip sebanyak ± 5µ l
2. Selanjutnya dengan menggunakan mikrotip, larutan DNA tersebut dimasukkan ke dalam jarum mikroinjeksi.
3. Capillary glass ditempelkan pada needle holder kemudian dimasukkan minyak mineral hingga penuh
4. Dengan bantuan capillary glass, minyak mineral dimasukkan ke dalam jarum mikroinjeksi hingga penuh, dengan memberi sedikit rongga antara larutan DNA dengan minyak mineral.
5. Langkah terakhir adalah capillary glass dilepas dari needle holder dan diganti dengan jarum mikroinjeksi yang telah berisi larutan DNA dengan minyak mineral. Jarum mikroinjeksi siap untuk proses penyuntikkan.
Lampiran7. Derajat Kelangsungan Hidup Embrio (DKHE) dan Derajat Penetasan (DP) pada ikan nila O.niloticus
1 30 26,67 16,67
2 30 40 26,67
rataan 30 33,33 21,67
simpangan baku 0 9,4 7,1
1 30 16,67 10
2 30 26,67 16,67
rataan 30 21,67 13,33
simpangan baku 0 7,1 4,7
1 30 70 63,33
2 30 86,67 80
rataan 30 78,33 71,67
simpangan baku 0 11,8 11,8
Jumlah telur yang diinjeksi Derajat Kelangsungan Hidup Embrio Derajat Penetasan
Keratin
HSC
Kontrol
Lampiran 8. Persentase embrio mengekspresikan transgen (PEMT) dan tingkat ekspresi Gen GFP pada ikan nila O.niloticus
