Gene delivery to Nile tilapia cells for transgenesis and the role of PI3K-c2α in angiogenesis.
Microinjection is commonly performed to achieve fish transgenesis; however, due to difficulties associated with this technique, new strategies are being developed. Here we evaluate the potential of lentiviral particles to genetically modify Nile tilapia cells to achieve transgenesis using three different approaches: spermatogonial stem cell (SSC) genetic modification and transplantation (SC), in vivo transduction of gametes (GT), and fertilised egg transduction (ET). The SC protocol using larvae generates animals with sustained production of modified sperm (80% of animals with 77% maximum sperm fluorescence [MSF]), but is a time-consuming protocol (sexual maturity in Nile tilapia is achieved at 6 months of age). GT is a faster technique, but the modified gamete production is temporary (70% of animals with 52% MSF). ET is an easier way to obtain mosaic transgenic animals compared to microinjection of eggs, but non-site-directed integration in the fish genome can be a problem. In this study, PI3Kc2α gene disruption impaired development during the embryo stage and caused premature death. The manipulator should choose a technique based on the time available for transgenic obtainment and if this generation is required to be continuous or not.
Cell Signaling &Nanobiotechnology Laboratory, Department of Biochemistry &Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil.|Nanocell Institute, Divinópolis, MG, Brazil.
Fernanda Maria Policarpo
FM
Tonelli
Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil.
Samyra Maria Dos Santos Nassif
SM
Lacerda
Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil.
Marcela Santos
MS
Procópio
Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil.
Breno Luiz Sales
BL
Lemos
Laboratory of Cellular Biology, Department of Morphology, Federal University of Minas Gerais, Belo Horizonte, Brazil.|National Institute for Amazonian Research (INPA), Manaus, AM, Brazil.
Luiz Renato
LR
de França
Cell Signaling &Nanobiotechnology Laboratory, Department of Biochemistry &Immunology, Federal University of Minas Gerais, Belo Horizonte, Brazil.|Nanocell Institute, Divinópolis, MG, Brazil.
Rodrigo Ribeiro
RR
Resende
D000072956:Adult Germline Stem Cells / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D000818:Animals; D030801:Animals, Genetically Modified; D023681:Cichlids / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000098:blood supply / Q000166:cytology / Q000378:metabolism; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005854:Germ Cells; D007814:Larva / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D008297:Male; D008845:Microinjections / Q000379:methods; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000235:genetics*; D019869:Phosphatidylinositol 3-Kinases / Q000172:deficiency / Q000235:genetics*; D014161:Transduction, Genetic / Q000379:methods*; D015053:Zygote / Q000254:growth & development / Q000378:metabolism
D019869:Phosphatidylinositol 3-Kinases