Transformation of Primary Cultures of Shrimp (Penaeus stylirostris) Lymphoid (Oka) Organ with Simian Virus-40 (T) Antigen

Abstract

Primary cultures of lymphoid (Oka) organ from Penaeus stylirostris were transformed with naked or Lipofectin-mediated pSV-3 neo, a shuttle vector containing the tumor (T) antigen gene from Simian virus-40. The transformed cells, OKTr-1 and OKTr-23, exhibited the following characteristics: rounded morphology forming grapelike aggregates, loosely adhesive, increased growth rate in Medium-199, resistance to G-418 (a neomycin analog marker in the shuttle vector), cloning efficiencies of 68.7% and 36.7% in soft agarose, respectively, and stability in liquid nitrogen storage. Immunofluorescence staining (IFA) of the transformed cells using a monoclonal antibody against SV-40 tumor antigen showed positive results. In contrast, primary cell cultures exhibited fibroblast-like morphology and formed a tight, adhesive monolayer on the surface of the culture vessel. They were sensitive to G-418, and showed negative results with IFA. To date, OKTr-1 and OKTr-23 have undergone 44 and 18 passages, respectively. Primary cultures of the lymphoid organ have not been successfully passaged beyond the primary stage.

Establishment of Shrimp Cell Lines: Perception and Orientation

“…Although the morphology and ultrastructure of WSSV have not been fully understood, several characteristics of this virus have emerged in recent years [71]. In addition to the effort on spontaneous transformation and immortalization by continuous maintenance and repeated passage of the cells in vitro and the 'organized neglect' [29] in the process of cell culture development, in the year 1995 researchers attempted to induce transformation in shrimp cells by transfection with oncogene [78]. Accordingly, in 2000 first transgenic expression in shrimp cells could be accomplished [73] followed by the development of vesicular somatitis virus-glycoprotein (VSV-G) pseudotyped retroviral vectors [37] and their successful integration in shrimp primary cell culture genome [36].…”

“…Considering the importance of inorganic salts for the maintenance of ionic balance and osmotic pressure [61], researchers have used KCl, MgSO 4 , MgCl 2 , and CaCl 2 at concentrations ranging from 0.9 to 3 g/l to supplement the required quantity in the growth medium [39,48,49]. To adjust osmolality, NaCl at a concentration ranging from 6 to 12 g/l [9,19,40,49] has also been added besides the balanced salt solutions [40,78]. Addition of vitamins [41,42], proline [49,56,57,82] and glutamine [28,82] has been proven to be the choice of supplements in the growth media.…”

“…Buffering agents such as HEPS [28,48,82] and NaHCO 3 have been incorporated by many researchers [19,28,48,49]. Growth factors such as epidermal growth factor at a concentration 20-30 ng/ml [51,63,78] and 10 units/ml of human recombinant interleukin-2 [78] have been used to improve the proliferation of cells in vitro. All these modifications have led to improvisation of growth media with enhancement in growth and multiplication of primary cell cultures, but have never lead to the establishment of any cell line.…”

“…Of the 50 selected publications, 17 reported (34 %) P. monodon [2-4, 7, 8, 10, 15, 23, 35, 41, 42, 44, 58, 69, 83, 89, 90] as the species of choice, eight researchers (16 %) used Penaeus japonicus [10,39,47,48,[55][56][57]72], seven (17 %) selected Penaeus chinensis [13, 19, 36-38, 40, 80] and Penaeus vannamei [18,26,27,49,51,63,82]. Moreover, six authors (16 %) selected Penaeus stylirostris [49,51,63,73,74,78] as the donor animal of tissues and organs. Besides, in two publications (4 %) Penaeus indicus [46,82] and Penaeus aztecus [18,64] were the species used.…”

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

“…It was found apparent that the lymphoid cells remained stable for longer period of time in the newly formulated SCCM with consistent growth and proliferation . Moreover, rapid monolayer formation, longevity and stability have been pointed out as the characteristics of lymphoid cells in culture (Nadala et al 1993), and accordingly the lymphoid tissue has been preferred over others for the development of shrimp cell culture (Chen and Kou 1989;Tapay et al 1995;Jose et al 2012;Jayesh et al 2012) and subsequent transformation into cell line .…”