title: Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer.
abstract: Genetically modified animals have numerous applications, ranging from basic research to livestock production and agriculture. Recent progress in animal cloning by nuclear transfer has made possible the production of transgenic animals using previously genetically modified cell lineages. However, to produce such lineages, an additional time for in vitro culturing and great manipulation is needed. Herein, we aimed to characterize different aspects of genetically modified cells compared to control cells, and we also analyzed the development rate of embryos produced by nuclear transfer by using them as nuclei donors after short or long periods of in vitro culturing (early versus late passages). We hypothesized that the genetic material inserted in the genome of these cells, associated with the prolonged time in culture, ultimately alters cell growth physiology and cell viability, which leads to impaired nuclei reprogramming potential and consequent reduction in the production of cloned blastocysts. Fetal fibroblasts expressing the enhanced Green Fluorescent Protein gene (eGFP) cultured for different periods in vitro were analyzed with respect to chromosomal numeric abnormalities, nuclear DNA fragmentation, the ratio of BAX and BCL2 gene transcripts, and the intensity of mitochondrial membrane potential, and they were then used as nuclei donors for somatic cell nuclear transfer (SCNT). Early passages were defined as fewer than 11 passages, and late passages were 18th passage (18(th)p) to 21(st)p. No differences were observed in the percentage of cells with chromosomal abnormalities or in the mitochondrial membrane potential analysis. eGFP cells in late passages and control cells in early passages were not different regarding DNA fragmentation; however, control cells in late passages presented higher fragmentation (P < 0.05). The Bax and Bcl2 gene expression ratio in control and transgenic cells presented different patterns regarding cell conditions during culture. For SCNT experiments, no difference was observed between groups reconstructed with early or late-passage cells when fusion (63.1% and 49%), cleavage (67.7% and 69.9%), eight-cell embryo (36.4% and 44.4%) and blastocyst (21.6% and 20.8%) rates were compared. In conclusion, culture behavior was different between control and eGFP cells. However, when different in vitro culturing periods were compared, long-term cultured transgenic fetal fibroblasts remained competent for blastocyst production when used as nuclei donors in the nuclear transfer technique, a feature needed for the genetic manipulation of cell culture experiments aiming for transgenic animal production.
authors.affiliation: Department of Veterinary Medicine, Faculty of Animal Sciences and Food Engeneering, University of São Paulo, São Paulo, Brazil. fabianabressan@usp.br
authors.forename: F F
authors.initials: FF
authors.lastname: Bressan
authors.forename: M S
authors.initials: MS
authors.lastname: Miranda
authors.forename: M C
authors.initials: MC
authors.lastname: Bajgelman
authors.forename: F
authors.initials: F
authors.lastname: Perecin
authors.forename: L G
authors.initials: LG
authors.lastname: Mesquita
authors.forename: P
authors.initials: P
authors.lastname: Fantinato-Neto
authors.forename: G F K
authors.initials: GF
authors.lastname: Merighe
authors.forename: B E
authors.initials: BE
authors.lastname: Strauss
authors.forename: F V
authors.initials: FV
authors.lastname: Meirelles
mesh_terms: D021581:Active Transport, Cell Nucleus; D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002417:Cattle; D019070:Cell Lineage; D002470:Cell Survival; D019976:Cloning, Organism; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D053652:Nuclear Transfer Techniques; D013997:Time Factors
chemical_list: C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins