INVESTIGADORES
RADRIZZANI HELGUERA Martin
artículos
Título:
Novel methods to induce exogenous gene expression in SCNT, parthenogenic and IVF preimplantation bovine embryos.
Autor/es:
FEDERICO PEREYRA-BONNET; ROMINA BEVACQUA; ISABEL LA ROSA; SIPOWICZ PABLO ; RADRIZZANI MARTIN; RAFAEL FERNANDEZ-MARTIN; SALAMONE DANIEL
Revista:
TRANSGENIC RESEARCH
Editorial:
SPRINGER
Referencias:
Lugar: Holanda; Año: 2011 vol. 20 p. 390 - 397
ISSN:
0962-8819
Resumen:
The import of exogenous DNA (eDNA)
from the cytoplasm to the nucleus represents a key
intracellular obstacle for efficient gene delivery in
mammalian cells. In this study, cumulus cells or
oolemma vesicles previously incubated with eDNA,
and naked eDNA were injected into the cytoplasm of
MII oocytes to evaluate their efficiency for eDNA
expressing bovine embryo production. Our study
evaluated the potential of short time co-incubation
(5 min) of eDNA with; (1) cumulus cells, to be used as
donor cells for SCNT and (2) oolemma vesicles
(vesicles) to produce parthenogenic transgene expressing embryos. In addition, we included a group
consisting of the injection of eDNA alone (plasmid)
followed by parthenogenic activation. Two different
pCX-EGFP plasmid concentrations (50 and 500 ng/ll)
were employed. The results showed that embryos
produced by SCNT and by vesicle injection assisted by
chemical activation were able to express the eDNA in
higher rates than embryos injected with plasmid alone.
The lower plasmid concentration allowed the highest
development rates in all groups. Using confocal
microscopy, we analyzed the interaction of FITClabeled eDNA with cumulus cells and vesicles as well
as oocytes injected with labeled plasmid alone. Our
images demonstrated that eDNA interacted with
cumulus cells and vesicles, resulting an increase in
its expression efficiency. In contrast, oocytes injected
with DNA alone did not show signs of transgene
accumulation, and their eDNA expression rates were
lower. In a further experiment, we evaluated if
transgene-expressing embryos could be produced by
means of vesicle injection followed by IVF. The lower
plasmid concentration (50 ng/ll) injected after IVF,
produced the best results. Preliminary FISH analysis
indicated detectable integration events in 1/5 of SCNT
blastocysts treated. Our studies demonstrate for the
first time that short term transgene co-incubation with
somatic cells can produce transgene-expressing mammalian SCNT embryos and also that parthenogenic,
eDNA- expressing embryos can be obtained by
injection of vesicles or eDNA alone. Moreover,
eDNA-expressing embryos can be also obtained by
cytoplasmic injection vesicles in IVF zygotes, simplifying the traditional IVF pronuclear injection
technique