neostop suppresses Hdh expression. (A) Northern analysis of total RNA from adult brain obtained from a wild-type (+/+), Hdh heterozygote (+/–) and HdhSTOP/+ (STOP/+)
mice is shown in the top panel. Hdh expression
in the HdhSTOP/+ brain
is equivalent to the expression in a Hdh+/– heterozygote.
The two major Hdh mRNA transcripts (differing at
their 3′ ends by the use of alternative
polyadenylation sites) are indicated with arrows. A region of the
RNA gel containing the 18S rRNA species (stained with ethidium bromide
to control for sample loading and indicated with an arrow on the
right) is shown in the bottom panel. (B) To determine
if any transcripts were produced from the HdhSTOP allele,
RT–PCR analyses were performed with total RNA isolated
from wild-type (+/+) or HdhSTOP/– (STOP/–)
E8.5 embryos using oligonucleotide primer pairs amplifying a region
within exon 1 [1], exons 3–5 [2] and
in the middle of the Hdh mRNA [3].
Reverse transcriptase was omitted from the cDNA synthesis reaction
to control for DNA contamination (–RT), while amplification
of β-actin cDNA was used as a control for
RNA integrity and cDNA synthesis (β-actin).
Note that PCR products corresponding to primer pairs 2 and 3 were
not detected by ethidium bromide staining using the HdhSTOP/– cDNA
template but were detected when using wild-type cDNA template. In
contrast, a PCR product corresponding to β-actin
was detected in equivalent amounts using the wild-type and HdhSTOP/– RNA
samples. A PCR product corresponding to a putative truncated HdhSTOP RNA [1] was
barely visible by ethidium bromide staining. (C)
Schematics of the Hdh wild-type mRNA transcript
and the putative truncated RNA transcript produced by the HdhSTOP allele
(HdhSTOP). The positions of the PCR
amplification products corresponding to the primer pairs 1, 2 and
3 are indicated by black rectangles.