INVESTIGADORES
RADRIZZANI HELGUERA Martin
artículos
Título:
Simple gene transfer technique based on I-SceI meganuclease and cytoplasmic injection in IVF bovine embryos
Autor/es:
BEVACQUA ROMINA; CANEL NATALIA ; HIRIART MI; SIPOWICZ PABLO; ROZENBLUM GUIDO TOMAS; VITULIO A; RADRIZZANI MARTIN; FERNANDEZ-MARTIN RAFAEL; SALAMONE DANIEL
Revista:
THERIOGENOLOGY
Editorial:
ELSEVIER SCIENCE INC
Referencias:
Lugar: Amsterdam; Año: 2013 vol. 80 p. 104 - 113
ISSN:
0093-691X
Resumen:
Transgenic methods in mammals remain inefficient. Recently, an easy and highly efficient transgenesis system using I-SceI meganuclease was described in Xenopus. The method consists of the injection into fertilized eggs of I-SceI reaction mixture with a plasmid DNA carrying the transgene flanked by the meganuclease recognition sites (pIS). In this work, the effect of I-SceI on gene transfer was tested for the first time in mammals, in particular the bovine. Different conditions were evaluated, including three pIS Pax6egfp concentrations and two pIS CAGegfp injection times (prior to IVM and post IVF). In addition, the quantity of transgene was measured by qPCR, and presence of transgene signals was evaluated by FISH. Transgene expression rates were higher for injection post IVF (79.1%, 91/115 and 63.0%, 75/119) than for injection prior to IVM (32.6%, 31/95 and 34.7%, 33/95), for the groups with I-SceI and without I-SceI respectively (P<0.05). Interestingly, injection with pIS plus I-SceI post IVF resulted inincreased frequency of non-mosaic transgene- expressing embryos (58.3%, 42/72 versus 29.7%, 25/84) for pIS plus I-SceI and pIS alone respectively (P<0.05), and FISH results confirmed that injection with I-SceI increased the proportion of embryos showing transgene signals in all of their blastomeres with respect to pIS alone (44.0%, 11/25 versus 6.9%, 2/29) for pIS plus I-SceI and pIS alone respectively (P<0.05). In addition,transgene copy number tended to be higher for the group injected with pIS plus I-SceIrespect to pIS alone. This work demonstrates that I-SceI gene transfer could be efficiently applied to the bovine, resulting in increased transgene signals.
