NDLI: Effects of long-term in vitro culturing of transgenic bovine donor fibroblasts on cell viability and in vitro developmental potential after nuclear transfer

Content Provider	Springer Nature Link
Author	Bressan, F. F. Miranda, M. S. Bajgelman, M. C. Perecin, F. Mesquita, L. G. Fantinato Neto, P. Merighe, G. F. K. Strauss, B. E. Meirelles, F. V.
Copyright Year	2013
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 Bcl$_{2}$ 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.
Starting Page	250
Ending Page	259
Page Count	10
File Format	PDF
ISSN	10712690
Journal	In Vitro Cellular & Developmental Biology - Animal
Volume Number	49
Issue Number	4
e-ISSN	1543706X
Language	English
Publisher	Springer-Verlag
Publisher Date	2013-03-22
Publisher Place	New York
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Bovine Cell culture Cell reprogramming Nuclear transfer Transgenesis Cell Biology Developmental Biology Stem Cells Cell Culture Animal Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Cell Biology Developmental Biology Medicine

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