Download Titel: Functional replacement of Gfi1 deficiency by Gfi1b obviously

Supplementary material, MS EMBO/2005/82698
third revision
Supplementary information
Materials and Methods
Generation of Gfi:Gfi1b and Gfi1:Gfi1P2A knock-in mice
Both knock-in mice were generated by modifying the previously described Gfi1:GFP knockin construct (Yücel et al., 2004). The constructs were transfected in R1 ES cells and selection
with G418 and ganciclovir was performed. Mice were held under specific pathogen-free
conditions in individually ventilated cages. All animal experiments were carried out according
to the German Animal Protection Law and were done under a license granted by the
Bezirksregierung Düsseldorf/NRW Germany (Nr.G022/02Z) and the Animal Care and Use
Committee of Tel Aviv University (M-04-014).
ES cells in which a homologous recombination event occurred were identified by Southern
blotting (supp. Fig. 1A and 2A) and were injected into mouse blastocysts to generate Gfi1 +/1b
or Gfi1P2A/P2A mutants. The functionality of the newly introduced Gfi1:Gfi1b knock-in allele
was confirmed by expression analysis of Gfi1 and Gfi1b in thymocytes (supp. Fig. 1B, 1C).
To exclude that remaining flox and frt sites affected the proper expression of the
Gfi1:Gfi1P2A knock-in allele, we prepared thymocytes from these animals, compared the
length of wild type and knock-in transcript by RT-PCR and sequenced it (data not shown).
Both transcripts had the same length and the knock in transcript had no other mutations except
the P2A exchange indicating that the transcription of the Gfi1:Gfi1P2A allele was not
disturbed (data not shown). Although it cannot be entirely ruled out that the remnant loxp and
frt sites interfere with gene regulation, it is unlikely that they play a major role in our
Gfi1:Gfi1P2A mutants since we detect the proper mRNA length and sequence.
Co-Immunoprecipitation
For coimmunoprecipitation, MEL 75A (clone 19) cells were used. After confluent growing,
the cells were lysed and cell lysates (300µg) were incubated with anti-Gfi1b (Santa Cruz
rabbbit polyclonal sc-22795) antibody overnight at 4 °C, followed by incubation with protein
A/G Agarose (Roche) for an additional 2 h. The pellet was washed three times with TBSTween buffer and once with PBS. Then the pellet was resuspended in SDS-containing buffer,
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Supplementary material, MS EMBO/2005/82698
third revision
heated at 95 °C, and centrifuged at 12,000 g. The supernatant was subjected to SDS-PAGE.
After transfer onto a nylon/nitrocellulose membrane, the samples were detected with either an
anti-Gfi1b (Santa Cruz goat polyclonal antibody sc-8559) or an anti-PIAS3 antibody (Santa
Cruz goat polyclonal sc-8154). For the input control, 15g protein was loaded on the gel.
RT-PCR and Quantitative Real-Time PCR
RNA from newborn inner ears was isolated using peqGOLD RNA pureTM (Peqlab). RNA was
treated with DNaseI (Promega) for 30 min. Reverse Transcription was performed using
Superscript II (Invitrogen) as described in the manufacturer`s protocol.
For
semiquantitative
RT-PCR
,
the
following
primers
were
used
5’-
CGCGGATCCATGCCACGGTCCTTTCTA-3’ and 5’-AGGTGTGTTTCTTCATGTCC-3’
for Gfi1b and 5’- GCGCTCATTCCTGGTCAAGA-3’ and 5’- TGTGTGGATGAAGGTGT
GTT-3’ for amplification of Gfi1.
Quantitative RT-PCR was performed in a 20 l reaction volume containing 900 nM of each
primer, 250 nM TaqMan probe, and TaqMan Universal PCR Master Mix (Applied
Biosystems). Reactions were monitored in an ABI PRISM 7700 Sequence Detection System
(Applied Biosystems). For detection of Gfi1 (Mm 00515853_m1) and Gfi1b (Mm
00492319_m1) Assays on Demand from Applied Biosystems were used. Every PCR was
performed in triplicates.
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Supplementary material, MS EMBO/2005/82698
third revision
Figure Legends
Supp. Figure 1
Genotyping and expression analysis of Gfi1:Gfi1b knock-in mice.
Heterozygous Gfi1:Gfi1b chimeras were born after injection of targeted ES clones in mouse
blastocysts and were mated with CMV-Cre transgenic mice to delete the floxed neomycin
selection cassette which was monitored by Southern blotting.
A
Southern blot analysis shows after digestion with BamHI and use of the 5' external
probe an 8 kb fragment from the wild type allele and a 14 kb fragment from the targeted allele
(left). Loss of the neomycin resistance gene can be detected using a SalI/SpeI double
digestion and the 3' probe. This leads to an 11.9 kb fragment from the wild type allele and a
5.6 kb fragment from the targeted allele unless the neomycin resistance gene was deleted. If it
is deleted, a 4.2 kb fragment is visible (middle and right).
B
Upper row: Gfi1 protein expression is detected in thymocytes from Gfi1+/+ and
Gfi1+/1b mice. Gfi11b/1b thymocytes have lost Gfi1 expression. Detection of -actin was used as
loading control. No expression of Gfi1 was detectable in thymocytes of homozygous
Gfi1:Gfi1b (Gfi11b/1b) knock-in mouse mutants, which was expected since homozygosity for
this allele disrupts the Gfi1 coding region. In contrast, Gfi1 expression is readily detected in
heterozygous Gfi1:Gfi1b knock-ins (Gfi1+/1b)
C
Detection of Gfi1b expression in thymocytes from Gfi1+/1b and Gfi11b/1b mice.
Expression of Gfi1b was undetectable in thymocytes of wild type animals (Gfi1+/+), as
expected, but was clearly present in Gfi1+/1b and at a significantly higher level also in Gfi11b/1b
animals. While these results confirm that functional Gfi1b alleles have been generated at the
Gfi1 locus, the relatively increased Gfi1b protein level in Gfi11b/1b thymocytes indicates a
regulatory phenomenon. It has been demonstrated that Gfi1 and Gfi1b are able to repress their
own promoters and in some cells can also mutually down modulate their expression by using
the same DNA binding sites in the Gfi1 and Gfi1b promoters, respectively (Doan et al., 2004,
Yücel et al., 2004, Vassen et al., 2005). It is therefore likely that the presence of one Gfi1 and
one Gfi1b allele in heterozygous animals results in different levels of expression than in
homozygous or wild type mice. This seemed to be the case at least for Gfi1b expression in
Gfi11b/1b cells.
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Supplementary material, MS EMBO/2005/82698
third revision
Supp. Figure 2
A
Genotyping of Gfi1:Gfi1P2A knock-in mice.
Southern blot of genomic DNA from ES cells (left) and mice (right) after HindIII
digestion. DNA fragments with a size of 15 kb originate from the wild type allele while the
targeted allele produces an 8 kb fragment.
B
Upper panel: Expression analysis by Western blot of whole cell extracts of thymocytes
from Gfi1+/+, Gfi1+/P2A, Gfi1P2A/P2A and Gfi1-/- mice. Lower panel: Uniform loading is
demonstrated by -actin expression level.
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Supplementary material, MS EMBO/2005/82698
third revision
Supp. Figure 3
A
Expression analysis of Gfi1:Gfi1b knock-in mice.
Semi-quantitative PCR from Gfi1+/+ and Gfi11b/1b inner ears of newborn mice. Upper
row: Gfi1 mRNA is not detectable in Gfi11b/1b inner ears. PCR with Gfi1b specific primers
shows a high mRNA expression in Gfi11b/1b inner ears compared to wild type (Gfi1+/+) mice.
Lower panel: Uniform loading of RNA is demonstrated by GAPDH- expression level.
B
Expression analysis by quantitative Real Time PCR from wild type (left) and Gfi11b/1b
(right) inner ears. PCR was performed with cDNA from newborn mice. The pictures show a
weak expression of Gfi1 and Gfi1b in Gfi1+/+ inner ears. In Gfi11b/1b inner ears no Gfi1 cDNA
can be detected, whereas the amount of Gfi1b cDNA is clearly increased in Gfi11b/1b inner
ears compared to the wild type inner ears.
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Supplementary material, MS EMBO/2005/82698
third revision
Supp. Figure 4
Lack of interaction between Gfi1b and PIAS3
Upper panel: Shown is the detection of PIAS3 in a Western Blot with MEL75A cell extracts
(input lane). MEL cell lysates precipitated with an anti Gfi1b antibody or an irrelevant anti
GST antibody were probed with an anti PIAS3 antibody but no Gfi1b/PIAS3 complexes
could be collected. As a control, the precipitates contain substantial amounts of Gfi1b when
probed with an anti Gfi1b antibody (lower panel) indicating a properly executed
immunoprecipitation reaction. Leftmost lane (lower panel) endogenous expression level of
Gfi1b in MEL75A cells.
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Supplementary material, MS EMBO/2005/82698
third revision
Table Legend
Table 1
Differential cell counts of wild type, Gfi1-/- , Gfi1P2A/P2A and Gfi11b/1b mice.
Differential cell counts from May-Grünwald-Giemsa stained blood smears and bone marrow
cytospins (after erythrocyte lysis). Shown are the mean values, their standard deviations and
the results of a two tail students t-test. Each value obtained from mutant mice was compared
to values obtained from wildtype animals. Significant results (p≤0.05) are marked with an
asterisk.
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