Period

2011 - 2014

Colaborators

Responsible: Raquel Pinho

PI: Dr.Luciana Relly Bertolini, University of Fortaleza, CE - Brazil

PI: Dr. Carlos Eduardo Ambrosio, University of Sao Paulo, SP - Brazil

Colaborator: Dr. James Murray, University of Californa, Davis, CA - United States

Colaborator: Dr. Elizabeth Maga, University of Californa, Davis, CA - United States

Colaborator: Dr. Kaio Cesar Tavares, University of Fortaleza, CE - Brazil

Colaborator: Dr. Marcelo Bertolini, University of Fortaleza, CE - Brazil

Colaborator: Dr. Leonardo Martins, University of Fortaleza, CE - Brazil

Location

• University of Fortaleza, CE - Brazil
• University of Sao Paulo, SP - Brazil
• University of California, Davis, CA - United States

Raquel Pinho’s main responsabilities

• Design and construction of DNA template for homologous direct repair of dystrophin gene in dogs using PCR for homology arms amplification.
• Design and assembly of pairs of Transcription Activator-Like Effector Nucleases (TALENS) targeting canine dystrophin gene by Golden Gate platform.
• Design and construction of siRNA targeting the Ku70 transcript.
• Silencing of the genes Ku70, ATP7IP and EP300 using siRNA in MDCK cells for enhanced gene editing of dystrophin gene by TALENs.

Description

The insertion of exogenous DNA into a host genome is achieved primarily through the use of DNA repair pathways such as Non-Homologous End Joining (NHEJ) and the Homologous Recombination (HR). The integration by NHEJ has a random feature and is much more common than HR insertions, which are more likely to produce gene targeting events . TAL effector nucleases (TALENs) and RNA interference (RNAi) can be used to increase the rate of specific integration and thus improving the efficiency of gene editing. In this work, we used short interference RNA (siRNA)-mediated gene silencing for transient inhibition of genes ATF7IP (implicated in histone methylation), EP300 (acetyltransferase) and Ku70 (essential to NHEJ) and a pair of TALENs RNAm complementary to canine muscle dystrophin (DMD) gene. MDCK I Canine Cells were transfected by lipofectamine 2000 (Invitrogen) with 320 pmol of siRNAs for ATF7IP and EP300; and 64 pmol of siRNA for Ku70 in different groups. After 40 hours cells were transfected with 15 µg of a vector derived from pEGFP- N1 (Clontech) containing two regions homologous to the canine DMD gene (left arm length: 873 bp and right arm length: 1370 bp) and 10 µg of TALEN mRNA. The cell selection was achieved with DMEM high glucose with 600μg/ml G418 for 14-16 days. The colonies collected through biopsies were analyzed by polymerase chain reaction and gene sequencing. Three pairs of primers were used; an endogenous control (GAPDH) , an internal control of the insert (Neo qPCR) and a primer set to confirm the occurrence of homologous recombination events (DMD3). .Groups showed great variation in cell death rate and consequently in the number of colonies: ATF7IP+Vector had highest number of colonies (648c) and the group EP300+Ku70+Vetor+TALENs the lowest one (1c) The highest rate of homologous recombination was in ATF7IP +Ku70+Vetor+TALENs group that had 40% of the neomycin positives cells confirmed as gene targeting events, a considerable increase in the recombination rate compared to the 3.1% in the control group transfected only with the template vector. That shows that the combined use of siRNAs and TALENs was a success for increasing directed gene editing events.

Publication

Use of RNAi–mediated gene silencing and Tal effector nucleases to enhance gene targeting events in dog cells. Master thesis (2014).