Ronna Hertzano – Karen B. Avraham's laboratory
hertzron@post.tau.ac.il
DIG in situ hybridization probes
Pou4f3 probe:
A 484 bp segment of the unique coding sequence of the Pou4f3 gene
(NM_138945
– nucleotides 13-496) was cloned into the pGEM T-easy
vector (Promega) for the generation of digoxigenin labeled sense and antisense
probes. Whole mount and section in situ hybridization on mouse cochleae were
performed as previously described, with minor modifications (Lanford et al., 2000;
Wu and Oh, 1996). RNA probes were transcribed from 2 µg of linearized plasmid
DNA that were either heat inactivated or cleaned by PCR column purification. RNA
probes were transcribed using the linearized DNA template, GTP, ATP and CTP at a
1 mM concentration (Roche), UTP 0.65 mM (Roche), Digoxigenin-11-uridine-5'triphosphate 0.35 mM (Roche), 1X transcription buffer (Roche), 100 units of
RNAguardTM (Amersham Pharmacia) and 20 units of T7 or SP6 RNA polymerase
(Roche). Transcription took place at 37ºC for 2 hours, followed by a 15 minute
digestion of the plasmid DNA by the addition of 20 units of DNAseI. The probes
were then run on a 1% agarose gel for quality control and precipitated over night with
an ammonium acetate RNA precipitation followed by a 75% ethanol wash. Probes
were stored in 75% ethanol at -80ºC until use. Whole mount in situ hybridization was
performed on wild type, Pou4f3ddl/ddl or Pou4f3-/- mice as indicated, using whole inner
ears that were dissected at the appropriate developmental time point and dehydrated
with a methanol gradient in PBS 0.1% Tween-20 (Sigma) and kept in methanol at 20ºC until use. Section in situ hybridization was performed on 12 µm cryostat
sections of whole inner ears of wild type, Pou4f3ddl/ddl or Pou4f3-/- mice that were
cryoprotected. All experiments were repeated at least three times with mice from at
least three separate litters.
For clone#3
Probe
Antisense
Sense
Cut with
SpeI
SalI
NdeI
SacI
BstXI
NsiI
AatII
SacII
Transcribe with
T7
SP6
Ronna Hertzano – Karen B. Avraham's laboratory
hertzron@post.tau.ac.il