INVESTIGADORES
HYON Sung Ho
congresos y reuniones científicas
Título:
CRISPR-ON system for the activation of the endogenous human lncRNA PLUTO
Autor/es:
CURTI LUCIA; GIMENEZ CARLA; GROSEMBACHER LUIS; HYON, SUNG HO; PEREYRA-BONNET FEDERICO
Lugar:
Mar del Plata
Reunión:
Congreso; LXIII Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2018
Institución organizadora:
Sociedad Argentina de Investigación Clínica
Resumen:
In order to improve the quality life of patients with Type 1 Diabetes some strategies are becoming to take part in alternative treatments. One of these is the development of cell therapies based on cellular reprogramming from stem cells or adult cells into insulin-producing cells. Our group has been working with CRISPR-ON system to switch on relevant human beta pancreatic cell genes. On the other hand non coding RNAs, microRNAs (miRs) and long non coding RNAs (lncRNAs), are starting to be a focus for this purpose. For example, the lncRNA PLUTO (HI-LNC71) influences interactions between an enhancer cluster and PDX1 (a key transcription factor in pancreas development). The objective of our work was to activate lncRNA PLUTO using CRISPR-ON system to improve future reprogramming protocols. For this, we designed four sgRNAs to target the PLUTO proximal promoter (-135/-50 pb respect to TSS) and cloned them into sgRNA expression vector (Addgene #47108). HEK293T cell line was transfected with dCas9-VP160 expression vector (Addgene #48226) and the 4 sgRNAs using Lipofectamine 2000® (Invitrogen). Six days after, RNA was extracted using miRNeasy Mini Kit (Qiagen). By RT-PCR and specific primers, our results showed that PLUTO was activated in CRISPR-ON group and no activation was observed in control group. In comparison with the positive control, human insulinoma, the product size suggests that an alternative splicing form was induced in HEK293T group. In conclusion, we could activate the lncRNA PLUTO using CRISPR-ON system in a model cell line. Further studies are needed to evaluate if this molecule has the capability of modulate PDX1 expression and become a new molecular tool in beta pancreatic cell differentiation protocols.
