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Universidade Estadual Do Ceará Pró-reitoria De Pós-graduação E Pesquisa Faculdade De Veterinária Programa De Pós-graduação Em Ciências Veterinárias
| Content Provider | Semantic Scholar |
|---|---|
| Author | Soares, Bruna Viana |
| Copyright Year | 2008 |
| Abstract | For animal transgenesis, several methods have been used to obtain offspring, however all of them have low efficiency. Therefore, new methodologies have been developed. In this scenario, cell penetrating peptides (CPPs) have already been tested as carriers of exogenous DNA. Crotamine, one of these CPPs, is a molecule obtained from the venom of rattlesnakes (Crotalus durissus terrificus) and has the ability to stably interact with nucleic acids by electrostatic interaction (forming a peptide-DNA complex). Therefore, the aim of this study was to verify the potential use of crotamine, in its native and linear forms, as exogenous DNA carrier molecule for the production of transgenic bovine embryos. For this, two studies were performed. In the first, embryos were exposed to native crotamine for different time intervals to evaluate its penetration. In addition, in vitro fertilized (IVF) embryos were exposed or microinjected with complex formed by native crotamine and reporter DNA for transfection evaluation. Concerning to penetration, images from confocal microscopy revealed that native crotamine was taken up by the embryos after 1 h of exposure. Regarding transfection, exposure and microinjection in the periviteline space with native crotamineDNA complex did not result in embryos expressing the transgene, whereas the intracytoplasmic DNA injection had higher rates of expression than intracytoplasmic native crotamine-DNA complex injection. In the second study, initially it was evaluated linear crotamine ability to complex DNA under different conditions, using native crotamine and lipofectamine as controls. Besides, we tested the embryotoxicity of linear crotamine by exposing bovine IVF embryos at different peptide concentrations for 6 h. To evaluate the efficacy of linear crotamine in inducing exogenous DNA expression, the linear crotamineDNA complex was microinjected into post-IVF zygotes. For complexation, the linear crotamine-DNA complex reached similar efficiencies at proportions 1:100 and 1:50 (DNA: peptide) and complexation was higher than controls for these groups. Therefore, we tested proportions up to 1:50 at different time intervals. Complexation occured very fast, since at least half of the final complexation (measured at 90 min) was achieved within initial 30 minutes for all groups. Regarding embryotoxicity, all embryonic development parameters recorded were similar compared to non-exposed embryos. Thus, zygote exposure had no effect on in vitro development. Regarding transfection, when using linear crotamine-DNA complex there is no increase in the efficiency of embryos expressing the reporter gene when compared to embryos microinjected with DNA alone. In this way, it is concluded that crotamine is able to translocate through the zona pellucida of bovine embryos. In turn, linear crotamine effectively forms a complex with DNA in the absence of detectable embryotoxicity under the tested conditions. Likely due to the high stability of complexation between the DNA molecules and these peptides, the use of both native and linear crotamine did not improve the expression of exogenous DNA in bovine embryos. |
| File Format | PDF HTM / HTML |
| Alternate Webpage(s) | http://www.uece.br/ppgcv/dmdocuments/IanaCampelo_Tese.pdf |
| Language | English |
| Access Restriction | Open |
| Content Type | Text |
| Resource Type | Article |
