INVESTIGADORES
RADRIZZANI HELGUERA Martin
congresos y reuniones científicas
Título:
INJECTION OF CELLS OR THEIR PARTS AFTER A SHORT EXPOSURE TO PLASMID CONSTRUCTS INDUCES TRANSGENESIS IN OVINE AND BOVINE EMBRYOS
Autor/es:
PEREYRA-BONNET FEDERICO; BEVACQUA ROMINA; GIBBONS A; CUETO M; FERNANDEZ-MARTIN RAFAEL; SIPOWICZ PABLO; RADRIZZANI MARTIN; SALAMONE DANIEL
Lugar:
Cordoba
Reunión:
Congreso; Annual Conference of the International Embryo Transfer Society; 2010
Institución organizadora:
International Embryo Transfer Society
Resumen:
As animal transgenesis is an essential tool in medicine and agriculture, it
is necessary to understand the mechanisms in order to develop novel methods of
transgenesis. We intended to determine if the injection of cells or their parts
into metaphase II (MII) oocytes after incubating with exogenous DNA can induce
transgenesis in embryos. Sperm cells for intracytoplasmic sperm injection (ICSI)
in ovine and cumulus cells for NT in bovine were incubated with pCX-EGFP plasmid
(5 to 50 ng μL-1) for 5 min in 2.8% Na citrate at 0°C before transfer
into a 10% polyvinylpyrrolidone (PVP) droplet and injection into MII oocytes
(previously enucleated in NT). In both species, oolemma-ooplasmic vesicles (OOV)
of 9 μm diameter obtained from MII oocytes by microsurgery were directly
incubated in PVP droplet with same pCX-EGFP concentration. As a control group,
pCX-EGFP suspension from PVP droplet was injected into MII oocytes. The NT
bovine zygotes were activated in 5 μM ionomycin (Io) for 4 min followed by 1.9
mM DMAP immediately for 3 h. In ICSI ovine, the
treatment with DMAP was applied 3 h later. Injected oocytes of OOV and controls
were activated as NT in bovine and as ICSI in ovine. Expression of EGFP was
determined with fluorescence microscopy under blue light (488 nm) at Days 4 to
7, and data were analyzed by Fisher test (P = 0.05). A group of NT, ICSI,
OOV, and control presumptive zygotes were treated with FITC-labeled bovine
fragments (100-300 bb) DNA in order to determine the binding sites with
exogenous DNA by laser confocal microscope analysis. Quantitative PCR (qPCR) was
performed to determine pCX-EGFP copy number at 0, 8, 16, and 24 h after Io in
all ovine treatments. Embryos expressing EGDP from all techniques were subjected
to FISH with rhodamine-labeled pCX-EGFP plasmid as a probe. In ovine, ICSI and
OOV injection green embryos at Day 4 [58% (61/105) v. 21.5% (8/38);
P < 0.05] and green blastocysts at Day 7 [71.8% (23/32) v.
66.6% (2/3)] were obtained, respectively. In bovine, green embryos [49.2%
(34/69) v. 29.7% (14/47); P < 0.05] and green blastocysts
[95.8% (23/24) v. 25.0% (2/8); P < 0.05] were produced by NT
and OOV injection, respectively. In controls, no green embryos were obtained in
ovine (0/47) and only low rates were observed in bovine [3.0% (2/65)]. Confocal
images of zygotes showed specific signal only in cumulus cells, spermatozoa, and
OOV The qPCR analysis showed similar plasmid copy number/zygote between
treatment and times in ovine (range 30 000-300,000). Embryo FISH images showed 1
to 2 specific signals in ICSI and NT interphases of both species and in OOV
ovine metaphases, the latter being direct evidence of transgene integration.
These results suggest that the injected cells or cellular parts (OOV)
dramatically increase transgenesis in ovine and bovine embryos. Until now, the
generation of NT and OOV embryos after short exposure to the DNA construction
has not been reported. We are performing embryo transfer and at the moment we
have a pregnancy derived from ICSI in ewes. In conclusion, the cellular
parts/transgene complex may affect exogenous DNA delivery or its interaction
with embryo DNA, facilitating the mechanism of transgenesis in mammals.