Attempts on producing lymphoid cell line from Penaeus monodon by induction with SV40-T and 12S EIA oncogenes.
In an attempt of in vitro transformation, transfection mediated expression of Simian virus-40 (T) antigen (SV40-T) and transduction mediated expression of Adenovirus type 12 early region 1A (12S E1A) oncogene were performed in Penaeus monodon lymphoid cells. pSV3-neo vector encoding SV40-T oncogene and a recombinant baculovirus BacP2-12S E1A-GFP encoding 12S E1A oncogene under the control of hybrid promoters were used. Electroporation and lipofection mediated transformation of SV40-T in lymphoid cells confirmed the transgene expression by phenotypic variation and the expression of GFP in co-transfection experiment. The cells transfected by lipofection (≥ 5%) survived for 14 days with lower toxicity (30%), whilst on electroporation, most of the cells succumbed to death (60%) and survived cells lived up to 7 days. Transduction efficiency in primary lymphoid cells was more than 80% within 14 days of post-transduction, however, an incubation period of 7 days post-transduction was observed without detectable expression of 12S E1A. High level of oncogenic 12S E1A expression were observed after 14 day post-transduction and the proliferating cells survived for more than 90 days with GFP expression, however, without in vitro transformation and immortalization. The study put forth the requirement of transduction mediated 'specific' oncogene expression along with telomerase activation and epigenetic induction for the immortalization and establishment of shrimp cell line.
National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, Kerala 682016, India; Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, Kerala 682016, India. Electronic address: jayesh@cusat.ac.in.
Jayesh
J
Puthumana
National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, Kerala 682016, India.
Priyaja
P
Prabhakaran
Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, Kerala 682016, India.
Rosamma
R
Philip
National Centre for Aquatic Animal Health, Cochin University of Science and Technology, Fine Arts Avenue, Kochi, Kerala 682016, India. Electronic address: isbsingh@gmail.com.
I S Bright
IS
Singh
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018274:Electroporation; D005786:Gene Expression Regulation; D008214:Lymphocytes / Q000502:physiology*; D009857:Oncogenes; D033561:Penaeidae / Q000235:genetics / Q000502:physiology*; D013539:Simian virus 40 / Q000502:physiology; D014161:Transduction, Genetic; D014162:Transfection
D000956:Antigens, Viral
12S E1A; In vitro transformation; Lymphoid cell culture; Oncogene; Penaeus monodon; SV40-T