title: Transfection of isolated rainbow trout, Oncorhynchus mykiss, granulosa cells through chemical transfection and electroporation at 12°C.
abstract: Over-expression or inhibition of gene expression can be efficiently used to analyse the functions and/or regulation of target genes. Modulation of gene expression can be achieved through transfection of exogenous nucleic acids into target cells. Such techniques require the development of specific protocols to transfect cell cultures with nucleic acids. The aim of this study was to develop a method of transfection suitable for rainbow trout granulosa cells in primary culture. After the isolation of rainbow trout granulosa cells, chemical transfection of cells with a fluorescent morpholino oligonucleotide (MO) was tested using FuGENE HD at 12 °C. Electroporation was also employed to transfect these cells with either a plasmid or MO. Transfection was more efficient using electroporation (with the following settings: 1200 V/40 ms/1p) than chemical transfection, but electroporation by itself was deleterious, resulting in a decrease of the steroidogenic capacity of the cells, measured via estradiol production from its androgenic substrate. The disturbance of cell biology induced by the transfection method per se should be taken into account in data interpretation when investigating the effects of under- or over-expression of candidate genes.
authors.affiliation: INRA LPGP UR037, Fish Physiology and Genomics, Campus de Beaulieu, Rennes Cedex, France. Electronic address: elisa.marivin@hotmail.fr.
authors.forename: E
authors.initials: E
authors.lastname: Marivin
authors.affiliation: INRA LPGP UR037, Fish Physiology and Genomics, Campus de Beaulieu, Rennes Cedex, France.
authors.forename: B
authors.initials: B
authors.lastname: Mourot
authors.affiliation: Inserm UMR S-991, Foie, Métabolismes et Cancer, Université de Rennes 1, Hôpital Pontchaillou, SynNanoVect Platform, Rennes, France.
authors.forename: P
authors.initials: P
authors.lastname: Loyer
authors.affiliation: INRA LPGP UR037, Fish Physiology and Genomics, Campus de Beaulieu, Rennes Cedex, France.
authors.forename: H
authors.initials: H
authors.lastname: Rime
authors.affiliation: INRA LPGP UR037, Fish Physiology and Genomics, Campus de Beaulieu, Rennes Cedex, France.
authors.forename: J
authors.initials: J
authors.lastname: Bobe
authors.affiliation: INRA LPGP UR037, Fish Physiology and Genomics, Campus de Beaulieu, Rennes Cedex, France.
authors.forename: A
authors.initials: A
authors.lastname: Fostier
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods*; D004958:Estradiol / Q000378:metabolism*; D005260:Female; D018014:Gene Transfer Techniques; D006107:Granulosa Cells / Q000166:cytology / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017686:Oncorhynchus mykiss / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids / Q000008:administration & dosage*; D011863:Radioimmunoassay; D014162:Transfection / Q000379:methods*
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004958:Estradiol
keywords: Electroporation; Granulosa; Primary culture of cells; Rainbow trout; Transfection