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Rafael Otero ¹, Darwin Hernandez ¹, Luiz Sergio de A Camargo ²

Affiliations
1 Universidad de Sucre - Campus Ciencias Agropecuarias, Sincelejo, CO
2 Embrapa Dairy Cattle Research Center, Juiz de Fora, MG, BR

Abstract

Objective: To evaluate the effect of treatment with trichostatin-A (TSA) on the production of bovine embryos, expressing the gene of the green fluorescent protein (GFP) generated by SCNT. Materials: 164 oocytes were distributed in three treatments, NT-GFP: newly reconstructed zygotes with genetically modified cells and not subject to TSA. NTTrico- GFP: newly reconstructed zygotes with genetically modified cells and subjected to TSA. PART: Zygotes generated by parthenogenetic activation, used as a control for the process of oocyte activation and culture of embryos. The rates of cleavage, blastocysts, and embryos that expressed GFP were assessed by contingency tables and chi-square tests. Results: The percentage of cleavage in the zygotes in the NT-GFP treatment was greater but did not vary significantly from the NT-Trico-GFP treatment. However, this last treatment had a higher percentage of blastocyst formation (p=0.077). The percentage of blastocysts from cleaved zygotes, the produced embryos were significantly higher (p<0.05) for the NT-Trico-GFP treatment than for the NT-GFP. In both treatments, all the blastocysts generated expressed the GFP protein. Conclusions: TSA improves the embryonic development of clones of genetically modified cattle that express GFP.

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Rafael Otero ¹, Darwin Hernandez ¹, Luiz Sergio de A Camargo ²

Affiliations
1 Universidad de Sucre - Campus Ciencias Agropecuarias, Sincelejo, CO
2 Embrapa Dairy Cattle Research Center, Juiz de Fora, MG, BR

Abstract

Objective: To produce bovine transgenic embryos by microinjection of a lentiviral vector with the eGFP gene as a marker. Methods: Four treatments were designed: T1=Control: fertilized in vitro (FIV) with cumulus-oocyte complexes (CCOs), cultivated in CR2 medium with 10% FBS and incubated at 38.5°C in an atmosphere of 95% humidity and 5% CO₂. T2=Control of culture medium: CCOs removed by vortex in the presence of hyaluronidase, FIV, grown in SOF medium in hermetic bag, with a gaseous mixture of 5% CO₂, 5% O₂ and 90% N₂ and humidity saturated at 38.5°C. T3=Microinjection control: CCOs removed microinjected with TALP medium, FIV and cultured under the same treatment conditions T2. T4=Microinjected with the lentivirus: CCOs removed microinjected with the lentiviral vector and FIV and cultured in the same conditions of the T2 and T3 treatments. The rate of development of blastocysts at day eight and the expression of the eGFP gene were evaluated. Findings: No significant statistical differences were found (p> 0.05) in the production of blastocysts at day eight, between treatments T1, T2, and T3. The percentage of blastocysts found in the T4 treatment was significantly lower (p <0.05) than in the other treatments. All embryos obtained in T4 expressed the transgene of interest. Application / Improvements: It is concluded that the culture conditions used were adequate for T1, T2 and T3, added that the microinjection with the lentiviral vector influences in some way the embryonic development, although, the technique was highly efficient for obtaining transgenic embryos.

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