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We have attempted to analyze the function of a maternal mRNA xlgv7 which is distributed as an animal-vegetal gradient in stage 6 oocytes using a combination of antisense oligodeoxynucleotide injection into oocytes followed by in vitro maturation and fertilization. Injection of 20 ng of the antisense oligodeoxynucleotide resulted in the destruction of the xlgv7 mRNA to undetectable levels. Upon maturation and fertilization the resulting embryos develop with no specific defects suggesting that the maternal store of xlgv7 in stage 6 oocytes is not required and that the embryo can develop solely with the maternal store of the xlgv7 protein. Also, these results demonstrate the feasibility of this approach in destroying a specific maternal RNA and assaying its effect on development.
Fig. 1. Gradient of xlgv7 mRNA distribution in stage 6
oocytes. Poly(A)+ RNA from total oocytes (lane 1), animal
pole (lane 2), middle region (lane 3), and vegetal pole (lane
4) was separated on formaldehyde-agarose gels, blotted,
and hybridized to random prime labeled xlgv7 cDNA and
Xenopus histone H4 probe. The arrow points to the xlgv7
mRNA and the H4 represents the histone H4 transcript.
Approximately 2 jig of RNA was loaded per lane except in
lane 1 in which a larger quantity (approximately 4jig) was
loaded.
Fig. 2. In situ hybridization with xlgv7 probe. Sagittal
sections of stage 6 oocytes hybridized to control (sense)
xlgv7 transcript (A); B and C show stage 2, 3 and 6 oocytes
hybridized with the antisense strand of xlgv7; D is a longer
exposure of C. Arrowheads mark the position of animal
pole in stage 6 oocytes. Magnification xl5 (darkfield).
Fig. 3. Northern blots and ethidium bromide staining of
total RNA (20 ng per lane) from stage 6 oocytes injected
with different amounts of sense and antisense
oligodeoxynucleotides. Panels A and B are different
exposures of the same blot hybridized to random labeled
xlgv7 probe. Lane 1, RNA from sham-injected control
oocytes; Lane 2, RNA from oocytes injected with 60 ng of
antisense no. 01; Lane 3, RNA from oocytes injected with
sense no. 1 oligonucleotide. Oocytes were incubated for 2h
post-injection. Panel B also shows the ethidium bromide
staining of the same gel before blotting. Panel C. Lane 1,
RNA from control sham-injected oocytes; Lane 2, RNA
from oocytes injected with 50ng of antisense; Lane 3, RNA
from oocytes injected with 10 ng of antisense
oligodeoxynucleotides no. 2.2. Panel C also shows the
ethidium bromide staining pattern of the gel before blotting
and the same blot re-probed with Xenopus histone H4
random labeled probe. The 18s refers to the position of 18S
rRNA on the gel.
Fig. 4. Hybridization of oligodeoxynucleotides to Northern
blot of poly (A)+ RNA from noninjected stage 6 oocytes.
Strips from two independent blots containing 20 ftg and 5/*g
of poly (A)+ RNA probed with antisense oligonucleotide
(01) showing specific hybridization to xlgv7 mRNA (arrow).
(S) Under the same conditions control sense oligonucleotide
(1) does not hybridize to oocyte poly (A)+ RNA. Similar
results were obtained with the 2.2 antisense probe.
Fig. 5. A and B. Northern blot of total RNA
(40^g per lane) from matured oocytes (A),
and 2-cell-stage embryos (B) originating from
the oocytes injected with 20 ng of
oligonucleotides. The blots were hybridized
with random labeled xlgv7 and Xenopus
histone H4 probes. Lanes Al and Bl, RNA
from control oocytes that were sham injected;
Lanes A2, 4 and B2, RNA from oocytes
injected with antisense (2.2) oligonucleotide;
Lanes A3 and B3, RNA from control oocytes
injected with sense (no. 1) oligonucleotide.
Arrow points the position of xlgv7 transcript.
Fig. 6. Maturation and development of oocytes injected with sense (S) and antisense (AS) oligodeoxynucleotides.
Fig. 7. Resynthesis of xlgv7 mRNA in embryos. Northern
blot of total RNA (25 /*g per lane) from stage 25 embryos
originating from oocytes injected with
oligodeoxynucleotides (20 ng per oocyte). The blot was
probed with xlgv7 and reprobed with Xenopus histone H4
random-labeled probes. Lane 1, RNA from sham-injected
embryos; Lane 2, RNA from oocytes injected with
antisense DNA (no. 2.2); Lane 3, RNA from embryos
injected with sense DNA (no. 1). Arrow marks the position
of xlgv7 transcript.
