Download Targeted disruption of the mouse E-Ras gene

Supplementary Methods
Digital Differential Display
We used the Digital Differential Display program
(http://www.ncbi.nlm.nih.gov/UniGene/info_ddd.shtml) to analyze gene representation
in cDNA libraries from ES cells and various tissues.
EST libraries derived from ES
cells were #10023, 500, 240, 1882 and 2512 (total 39323 entries).
Libraries used to
represent various somatic tissues were #8901, 8902, 9741, 539, 598, 9978, 9980, 8621,
10030, 10402, 553, 519, 544, 7240, 10407, 5400, 12252, 5369, 9938, 1140, 9956, 2518,
2602, 2509, 2601, 5389, 4042, 9950, 2590, 2554, 9946, 4263, 2523, 12248, 509, 9974,
9959, 12249, 5497, 1771, 868, 5403, 497, 8907, 7222, 1870, 1783, 470, 2520, 5390,
2526, 2527, 1876, 5423, 5361, 9847, 1878, 2579, 9839, 5368, 1879, 3780, 5352, 5357,
12267, 12265, 12266, 7256, 12078, 12309 and 498 (total 625654 entries).
Primer sequences
45328-AS1 (5’- CCCATGTTACCACGTAACTT-3’)
45328-race11 (5’- GGTAACTTGGTCGGAAAGGGAAAT-3’)
ERas-gw-S (5’-AAAAAAGCAGGCTGGGGAATGGCTTTGCCTA-3’)
ERas-gw-AS (5’-AGAAAGCTGGGTCAAAGATCTTCAGGCTACAG-3’)
ERas-gw-AS2 (5’-AGAAAGCTGGGTCATTTCTGGTGTCGGGTC-3’)
PI3K-delta-gw-U (5’- AAAAAGCAGGCTCCATGCCCCCTGGGGTGGACTGCC-3’)
PI3K-delta-gw-L (5’-AGAAAGCTGGGTCTACTGTCGGTTATCCTTGGAC-3’).
hyg.upper (5’-AAAAAGCAGGCTACCATGAAAAAGCCTGAACTCACC-3’)
hyg.lower (5’-AGAAAGCTGGGTCTATTCCTTTGCCCTCGGACGAGTG-3’)
mutS (5’-CTTGCAGGATCGCCTAGGCTCCGGGCGTAT-3’)
mut AS (5’-ATACGCCCGGAGCCTAGGCGATCCTGCAAG-3’).
hygTopII.HpaI (5’-GTTAACCCAAAGATGAAAAAGCCTGAACTCACC-3’)
hygLast.XbaI (5’-TCTAGACTATTCCTTTGCCCTCGGACGAGT-3’)
ERAS-S118 (5’-ACTGCCCCTCATCAGACTGCTAC-3’)
ERAS-AS264 (5’-ACTGTGCCCAAGCCTCGTGACTTT-3’)
ERAS-U527 (5’-CTGGTGATGGTGTGCTGGGCGTCT-3’)
ERAS-S812 (5’-CGAATCAAGCAAGAAGACCCGACA-3’)
-geo screening 1 (5’-AATGGGCTGACCGCTTCCTCGTGCTT-3’)
ERAS-screening1 (5’-GGGAGGGAGGGCAAGGGCAGAGGGCT-3’)
ERAS-TW1 (5’-CTCAAGAAAGTCCGCTTCCCGCTCAG-3’)
ERAS-TW2 (5’-GGAACGCCAGAGCCCTGCTTACCTGT-3’)
E-Ras3’probe-s (5’-AGCTGGAGCGTCCGGGTCATCGTC-3’)
E-Ras3’probe-as (5’-AGGCTGGGAATTAAAGGCGTGAAC-3’)
hHRAS2-S (5’-GATCAGCACACAATAGGCAT-3’)
hHRAS2-AS (5’-ACTCCCACCCACACAACACT-3’)
Ha-ras2-gw-s (5'-CACCATGGAGCTGCCAACAAAGCC-3')
Ha-ras2-gw-as (5'-TCAGGCCACAGAGCAGCCACAGTG-3')
B-Raf-gw-s (5'-CACCTATAAGATGGCGGCGCTGAGC-3')
B-Raf-gw-as (5'-TCAGTGGACAGGAAACGCACCATATC-3')
Construction of entry vectors for Gateway cloning system
EcoRI/BamHI fragments of pCMV-Ras, pCMV-RasV12 and pCMV-RasN17 (Clontech)
were inserted into the same site of pENTR-1A (Invitrogen) to construct pENTR-HRas,
pENTR-HRasV12 and pENTR-HRasN17. Coding region of mouse ERas was amplified
by RT-PCR from ES-cell derived total RNA with primers ERas-gw-S and ERas-gw-AS.
Coding region of mouse ERas lacking the CAAX motif was amplified by RT-PCR from
ES-cell derived total RNA with primers ERas-gw-S and ERas-gw-AS2. Coding region
of mouse PI3kinse p110 was amplified from an EST clone (#4192906) with primers
PI3K-delta-gw-U and PI3K-delta-gw-L. After adding the attB1 and attB2
recombination sites at both ends by additional PCR with primers attB1 and attB2, we
inserted these PCR products into pDONR201 (Invitrogen) with BP recombinase to
construct pDONR-mERas, pDONR-mERasC and pDONR-PI3K. Coding regions of
human BRaf and hERas were amplified with primers BRaf-gw-s/as and
Ha-ras2-gw-s/as from human testis cDNA and pCR2.1-hERas.
PCR products were
inserted into pENTR/D-TOPO (Invitrogen) by TA cloning to construct pENTR-BRaf
and pENTR-hERas.
Construction of destination vectors for Gateway systems
We constructed pCAG-IRES-neo by replacing the NdeI/EcoRI fragment of pIRES-neo
(Clontech), which contained the CMV promoter, with the NdeI/EcoRI fragment of
pCX-EGFP, which contained the CAG promoter.
Gateway refA Cassette (Invitrogen)
was blunt-ligated into the EcoRI site of pCAG-IRES-neo to construct
pCAG-gw-IRES-neo. Gateway refA Cassette was blunt-ligated into the XhoI site of
pCAG-IP to construct pCAG-IP-gw.
Two oligonucleotides , oligo-HA-s and
oligo-HA-as were annealed and inserted into the EcoRI/BamHI site of pCAG-IRES-neo
to construct pCAG-HA-IRES-neo.
Gateway refB Cassette was blunt-ligated into the
BamHI site of pCAG-HA-IRES-neo to construct pCAG-HA-gw-IRES-neo. An
SpeI/XhoI fragment of pCAG-HA-gw-IRES-neo was inserted into the SpeI/XhoI site of
pCAG-IP to construct pCAG-IP-HA-gw.
pCAG-IP-myc-gw was constructed as
described elsewhere (Y. Tokuzawa et al., Mol. Cell. Bio., in press). pCAG-gw-IH was
constructed as follows: cDNA of hygromycin resistance gene was amplified from
pHPCAG with primers hyg.upper and hyg.lower. Additional PCR with primers attB1
and attB2 was performed and PCR product was inserted into pDONR201 to construct
pDONR-hygro. The SacII site in the hygromycin resistance gene was mutated without
changing encoded amino acid (CCGCGG to CCTAGG) by QuickChange Site-Directed
Mutagenesis Kit (Stratagene) to construct pDONR-hygromut. Primers used were mutS
and mut AS. cDNA of the mutated hygromycin resistant gene was amplified from
pDONR-hygromut with primers hygTopII.HpaI and hygLast.XbaI and ligated into
pCR2.1. An HpaI/XbaI fragment of this plasmid was ligated into the SmaI/XbaI site of
pIRES-neo (Clontech) to construct pIH. An SpeI fragment of pIH was replaced with an
SpeI fragment of pCAG-gw-IRES-neo containing the CAG promoter and Gateway
cassette to construct pCAG-gw-IH.
Construction of retroviral vectors
An SalI/EcoRI fragment of pCX-EGFP was blunt-ligated into the MluI/EcoRV site of
pIRESpuro (Clontech) to construct pCAG-IP. An EcoRI/BamHI fragment of
pCMV-HRasV12 was introduced into the EcoRI/BamHI site of pCAG-IP to construct
pCAG-HRasV12-IP. An EcoRI/XbaI fragment of pCAG-HRasV12-IP was blunt-ligated
into the BamHI/EcoRI site of pMX to construct pMX-HRasV12-IP. An EcoRV/XbaI
fragment of pIP was blunt-ligated into the BamHI/SalI site of pMX to construct pMX-IP.
Gateway refB Cassette was blunt-ligated into the BamHI site of pMX-IP to construct
pMX-gw-IP. pMX-mERas-IP and pMX-hERas-IP were constructed by the Gateway
technology.
Construction of targeting vector
A bacterial artificial chromosome (BAC) clone containing the mouse E-Ras gene was
identified by colony hybridization with the full-length E-Ras cDNA as a probe. A DNA
fragment containing intron 1 was amplified from the BAC clone by PCR with primers
ERAS-S118 and ERAS-AS264. An amplified fragment (3.3 kb) was used as the
5’-homologous region of a targeting vector that replaces exon 2 with a -geo cassette.
To make the 3’-homologous region, we obtained 3’-flanking region with TOPO Walker
Kit (Invitrogen). Primers used were ERAS-U527 for primer extension and ERAS-S812
for PCR. A 1.84-kb fragment obtained from a PstI-digested DNA pool was used as the
3’- homologous region. The resulting targeting vector was linearized with SacII and
introduced into RF8 ES cells by electroporation.
PCR screening for clones with correct homologous recombination
G418-resistant colonies were screened by PCR with primers -geo screening 1 and
ERAS-screening1. PCR was performed with Expand Long Template PCR System
(Roche). The PCR Program consisted of an initial denature at 94 °C for 10 sec., 36
cycles of 94 °C for 5 sec., 55 °C for 5 sec. and 68 °C for 3 min. and an additional
extension at 68 °C for 20 min. Specific amplification with these primers yielded a
2.6-kb product from correctly targeted clones.
Preparation of probes for Southern hybridization
To produce the 5' probe, we generated a DNA fragment from the 5'-flanking region with
TOPO Walker Kit. Primers used were ERAS-TW1 for primer extension and
ERAS-TW2 for PCR. A 4-kb fragment amplified from an NsiI-digested DNA pool was
inserted into pCR2.1. A 0.7-kb EcoRI fragment of this plasmid was used as the 5 probe.
The 3 probe was amplified from the BAC clone with primers E-Ras 3probe-s and
E-Ras 3probe-as.
Construction of the expression vector for activated PI3 kinase
An MluI/EcoRV fragment of pIH was replaced with an MluI/EcoRI fragment of
pCAG-IRES-neo containing the CAG promoter to construct pCAG-IH. A
HindIII/EcoRV fragment of pUSEamp-myr-p110 (Upstate Biology) was treated with
Taq polymerase and subcloned into pCR2.1 by TA cloning. A BamHI fragment of this
plasmid was subcloned into the same site of pCAG-IH to produce pCAG-myr-p110-IH
AP1 enhancer reporter assays
pCAG-IP, pCAG-IP-ERas, pCAG-IP-HRasV12, or pCAG-IP-HRasN17 was introduced
into MG1.19 ES cells along with pAP1-TAL (Clontech), in which a firefly luciferase
(FL) is driven by a minimum thymidine kinase promoter and AP1 enhancer. To
monitor transfection efficiency, pRL-TK (Promega), in which Renilla luciferase (RL) is
driven by a thymidine kinase promoter was co-transfected. After a 24-hour culture in
medium devoid of FBS, cell lysates were collected and luciferase activities were
measured with Dual Luciferase Reporter assay System (Promega).
Legend for Supplementary figures
Figure S1
a. pMX-IP vector. LTR, long terminal repeat; 
packaging signal; MCS, multiple
cloning site; IVS, intron; IRES, internal ribosome entry site; puro, puromycin resistant
gene
b. Flowcytometry analysis of cells transfected with pMX-IP or pMX-EGFP-IP.
Figure S2
a. Interaction of with BRaf. MG1.19 cells were transfected with myc-hERas (1),
myc-mERas (2), myc-HRas (3), myc-HRasV12 (4), myc-HRasN17 (5), or EGFP (6),
along with HA-BRaf. Twenty-four-hours later, cell lysates were collected and blotted
with  -myc antibody (bottom) and -HA antibody (middle). Lysates were then purified
with agarose-conjugated -myc antibody and precipitates were blotted with -HA
antibody (upper).
b. Effect of ERas and HRas on the AP1 enhancer reporter construct. Shown are averages
and standard deviations of normalized luciferase activity from four independent
experiments. *, P < 0.05; **, P < 0.01, compared to mock (pCAG-IP).
c. Interaction with endogenous ERas and PI3K p85. RF8 ES cell lysates were
precipitated with -p85 antibody (#06-195, Upstate Biotechnology) or normal rabbit
IgG. Precipitates were separated by SDS-PAGE, transferred to a membrane, and blotted
with -ERas antiserum.
d. Akt phosphorylation in ERas-null ES cells. ES cells described in fig. 3f were grown
on gelatin-coated plates in the medium supplemented with LIF. Lysates from log-phase
growing cells were analyzed by Western blotting with -phospho-Akt (Ser 473) or
-Akt antibody (#9270, New England Biolabs).
e. Inhibition of growth promoting effect of ERas by LY294002. NIH 3T3 Cells
transfected with parent pMX vector (mock), pMX-hERas, -mERas, myrP110, or
HRasV12 were plated at 5000 cells per well in 24-well plates and cultured with DMSO
(vehicle), LY294002 (10
M) or PD098059 (50
M). Cell numbers were determined
after 10 days. **, P < 0.01, compared to mock (n=4).