Use of integrases to target genetic parts resulting in gene regulation in eukaryotic cells
Mayna Gomide (1,2), Thais Torquato (1,2), Leila Barros (1), Luciana Carvalho (3), Mariana Almeida (1), Rayane Lima (1), Marco Oliveira (1), Lilian Florentino (1), Andre Murad (1), Martin Bonamino (3), Cintia Coelho (1,2) and Elibio Rech (1)
1- Brazilian Agricultural Research Corporation - Embrapa, Brazil; 2- University of Brasília – UnB, Brazil; 3- National Cancer Institute – INCA, Brazil, Correspondence: elibio.rech@embrapa,br
DNA recombinant technology was a landmark for the development of the synthetic biology, which constitutes an intersection area between biology and engineering and should contribute to designing biological systems. The development of effective tools, which allow us to increase precision on gene regulation are essential for proper genetic manipulation. Current information has demonstrated effective control of the RNA polymerase flux using different integrases, capable to catalyse unidirectional inversion of DNA to turn on/off regulatory genes in prokaryotic cells. However, knowledge about the functionality of integrases in eukaryotic cells is still limited. Here we show the remarkable functional capability of bacteriophages serine integrases in plant, animal and human cells. A co-transformation plasmid system was utilized to evaluate integrases in A. thaliana protoplasts, bovine fibroblast and HK293T cells. The first plasmid contained the codon optimized integrase gene sequences under constitutive promoters. The second plasmid was the reporter plasmid that contains the gfp coding sequence gene in reverse complement orientation and flanked by the attB and attP sites of each integrase under constitutive promoter. Once the integrases were expressed, it would flip the coding sequence to its correct orientation and promoting GFP expression. The results proved that the coding sequence was flipped leading to the RNA polymerase flux through the DNA molecule at the forward orientation and the GFP expression, which was detected by fluorescent microscopy and flow cytometry. The coding sequence inversion was detected by PCR and sequencing analyses. We anticipate our results to be an initial point for development of more complex models of gene regulation in eukaryotic cells using integrases.