Please use this identifier to cite or link to this item: http://ir.swu.ac.th/jspui/handle/123456789/12749
Title: Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon)
Authors: Thansa K.
Rungsiwiwut R.
Kitiyanant N.
Taengchaiyaphum S.
Keywords: biological marker
chemical compound
enhanced green fluorescent protein
hyalin
plasmid DNA
protein
complementary DNA
enhanced green fluorescent protein
green fluorescent protein
animal cell
animal experiment
Article
autofluorescence
blood cell
cell count
cell death
cell immortalization
cell line
cell proliferation
cell viability
controlled study
Cytomegalovirus
electroporation
female
gene construct
gene expression
genetic transfection
in vitro study
in vivo study
lipofection
male
nonhuman
nonviral gene delivery system
Penaeus monodon
plasmid
primary cell
priority journal
process optimization
promoter region
protein expression
reverse transcription polymerase chain reaction
signal transduction
Western blotting
White spot syndrome virus
animal
blood cell
electroporation
gene transfer
genetics
HEK293 cell line
human
immediate early gene
Penaeidae
virus gene
Animals
Cell Line
Cytomegalovirus
DNA, Complementary
Electroporation
Female
Gene Transfer Techniques
Genes, Immediate-Early
Genes, Viral
Green Fluorescent Proteins
HEK293 Cells
Hemocytes
Humans
Male
Penaeidae
Plasmids
Promoter Regions, Genetic
Issue Date: 2018
Abstract: To achieve in creating permanent shrimp cell lines, cellular arrest of primary cells in the culture is needed to be firstly solved. Considering the insertion of some markers affecting cellular proliferation into primary haemocytes in order to produce the black tiger shrimp cell line and the very low percent of transduced cells previously reported in penaeid shrimps, these paved us the way to set up suitable gene delivery protocols to increase percent of transduced cells in the shrimp as our primary aim. In this study, electroporation and lipofection were used to transfer construct plasmids (pLL3.7 plasmids containing CMV promoters and pGL-IE1-126(A)-EGFP plasmids carrying WSSV IE1 promoters) into primary haemocytes. As it was difficult to distinguish between cells expressing EGFP signal and auto-fluorescence of many dead cells occurred by electroporation during the first 72 h of experiment; so, only lipofection was managed to deliver plasmids into primary cells. Surprisingly, numbers of suspected proliferative cells were derived after electroporation with no insertion of immortalising markers. These cells survived in vitro for up to 45 days with high rate of cell viability, but the number of viable cells decreased throughout the experiment. In addition, these cells expressed genes and proteins closely related to hyaline cells determined using RT-PCR and western blot. For the lipofection experiment, no green fluorescence signal was detected in any primary cell introduced with these plasmids, suggesting that plasmids were not successfully inserted into cells. Also, a number of primary haemocytes had the apoptotic cell death characteristic within 5 days after lipofection. These possibly result from using inappropriate lipofection protocol and chemical substances. In summary, finding out suitable protocols to elevate the percent of transduced cells is still necessary. Additionally, continuous shrimp cell lines would be possibly established by transforming suspected proliferative cells derived from electroporation in this study. © 2018 Elsevier Ltd
URI: https://www.scopus.com/inward/record.uri?eid=2-s2.0-85049844332&doi=10.1016%2fj.fsi.2018.07.030&partnerID=40&md5=d69f508c481d14082a7c525f4db3bc64
http://ir.swu.ac.th/jspui/handle/123456789/12749
ISSN: 10504648
Appears in Collections:SCOPUS 1983-2021

Files in This Item:
There are no files associated with this item.


Items in SWU repository are protected by copyright, with all rights reserved, unless otherwise indicated.