40
General Methods
Equipment
Biologic
materials
Chemicals
However, recent progress in gene technology has enabled the immortalization of cells by the introduction of oncogenes. Tapay et al. (1995) obtained a continuous cell line from shrimp by transforming primary cultured lymphoid cells with simian virus (SV)-40 (T) antigen. Their method
is as follows.
Materials
- Dissecting tray
- 6-well plastic plate (Falcon Primaria 25 cm 2 ; Becton Dickinson
Labware)
- Primary cultures oflymphoid (Oka) gland cells from 50-60 g shrimp
(Penaeus stylirostris; prepared by the method described in chapter
12, subprotocol 7)
- Plasmid DNA pSV-3 Neo (an 8.6 kb construct containing gene sequences from SV-40 large tumor antigen (T-antigen»
- pBR 322
- Antibiotic resistance markers G-418
- Epidermal growth factor (EGF) from the mouse
- Human recombinant interleukin (IL)-2 (Calbiochem)
- Streptomycin
- Penicillin
- Ampicillin
- Geneticin 418 (Sigma)
- Lipofectin (Gibco BRL)
Solutions - Shrimp extract
Suspend shrimp heads in double-strength Leibovitz's L-15 medium
(10 mllg tissue) and homogenize.
Centrifuge the homogenate at 3,000 g for 30 min, and then centrifuge
the supernatant again at 10,000 g for 50 min.
Centrifuge the resulting supernatant at 330,000 g for 1 h.
Pass the extract through a membrane filter.
- MediumD
Supplement Leibovitz's L-15 medium (2 x) with 20% FBS, 8% shrimp
extract and 20 ng/ml EGF from the mouse, 10 units/ml human recombinant IL-2, 100 mg/ml streptomycin, and 100 IU/ml penicillin.
General Methods
Equipment
Biologic
materials
Chemicals
However, recent progress in gene technology has enabled the immortalization of cells by the introduction of oncogenes. Tapay et al. (1995) obtained a continuous cell line from shrimp by transforming primary cultured lymphoid cells with simian virus (SV)-40 (T) antigen. Their method
is as follows.
Materials
- Dissecting tray
- 6-well plastic plate (Falcon Primaria 25 cm 2 ; Becton Dickinson
Labware)
- Primary cultures oflymphoid (Oka) gland cells from 50-60 g shrimp
(Penaeus stylirostris; prepared by the method described in chapter
12, subprotocol 7)
- Plasmid DNA pSV-3 Neo (an 8.6 kb construct containing gene sequences from SV-40 large tumor antigen (T-antigen»
- pBR 322
- Antibiotic resistance markers G-418
- Epidermal growth factor (EGF) from the mouse
- Human recombinant interleukin (IL)-2 (Calbiochem)
- Streptomycin
- Penicillin
- Ampicillin
- Geneticin 418 (Sigma)
- Lipofectin (Gibco BRL)
Solutions - Shrimp extract
Suspend shrimp heads in double-strength Leibovitz's L-15 medium
(10 mllg tissue) and homogenize.
Centrifuge the homogenate at 3,000 g for 30 min, and then centrifuge
the supernatant again at 10,000 g for 50 min.
Centrifuge the resulting supernatant at 330,000 g for 1 h.
Pass the extract through a membrane filter.
- MediumD
Supplement Leibovitz's L-15 medium (2 x) with 20% FBS, 8% shrimp
extract and 20 ng/ml EGF from the mouse, 10 units/ml human recombinant IL-2, 100 mg/ml streptomycin, and 100 IU/ml penicillin.