Publication:
Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon)

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Issued Date

2018

Resource Type

Article

File Type

application/pdf

ISSN

10504648

DOI

10.1016/j.fsi.2018.07.030

Other identifier(s)

2-s2.0-85049844332

Rights Holder(s)

มหาวิทยาลัยศรีนครินทรวิโรฒ

Bibliographic Citation

Fish and Shellfish Immunology. Vol 81, No. (2018), p.204-213

Suggested Citation

Thansa K., Rungsiwiwut R., Kitiyanant N., Taengchaiyaphum S. Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon). Fish and Shellfish Immunology. Vol 81, No. (2018), p.204-213. doi:10.1016/j.fsi.2018.07.030 Retrieved from: https://hdl.handle.net/20.500.14740/5837

Title

Optimisation of electroporation and lipofection protocols to derive the black tiger shrimp cell line (Penaeus monodon)

Author(s)

Thansa K.
 Rungsiwiwut R.
 Kitiyanant N.
 Taengchaiyaphum S.

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

Subject(s)

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

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URI

https://hdl.handle.net/20.500.14740/5837

Collections

Scopus: Year 1983-2021

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