Supplemental methods
Construction of Foxc2 targeting vector
We adopted a recombination system utilizing homologus recombination in E. coli to
construct a huge Foxc2 conditional targeting vector (21kb). E. coli containing a BAC
clone encoding Foxc2 gene and its promoter region (#RP23-214P21, DNA source:
C57BL/6L kidney & brain, BACPAC Resources Center at Children's Hospital Oakland
Research Institute, Oakland, CA) was expanded and transformed with pRed/ET (Gene
Bridges
GmbH,
Heidelberg,
Germany)
by
electroporation
according
to
the
manufacture’s instruction. To insert 5’-loxP site, floxed PGK/Tn5-neo-pA cassette in
pPGK-neo loxP vector (Gene Bridges GmbH) was PCR amplified (KOD FX, Toyobo,
Osaka, Japan) by primers ‘a’ and ‘b’. A 52 bp homology arm of corresponding genome
sequence was added to each primer and a KpnI site was introduced to primer ‘a’ (Fig. S1,
Step I, Table S1). The amplified PCR product was gel purified and electropolated into
the pRed/ET transformed E. coli carrying the BAC clone in which Red/ET proteins were
induced by L-arabinose to facilitate homologous recombination (Step II). Then,
5’-upstream homology arm and 3’-downstream homology arm (0.3 kb each) were PCR
amplified by primers ‘c’ to ‘f’, and subcloned into SfiI sites of pAEF vector to retrieve
Foxc2 genome with a inserted loxP from the BAC clone (Step III, 6). The Foxc2
retrieving vector was linearized with AscI digestion and electroporated into the E. coli
carrying the 5’-loxP inserted BAC clone after the induction of Red/ET (Step IV). After
kanamycin and ampicillin selection, Foxc2 5’-loxP vector was isolated from a surviving
colony, and treated with Cre recombinase (New England Biolabs, Ipswich, MA, Step V)
to remove the PGK/Tn5-neo-pA cassette adjacent to the 5’-loxP site. After removal of the
PGK/Tn5-neo-pA cassette, the retrieved Foxc2 5’-loxP vector was electroporated into a
recombineering host strain, SW102 (National Cancer Institute at Frederick, Frederick,
MD). To generate second loxP site, homology arms (0.3 kb each) containing
3’-untranslated region of Foxc2 gene were PCR amplified by primers ‘g’ and ‘j’ and
subcloned into SfiI sites of pAHV (Step VI, 6) to generate Foxc2 3’-loxP inserting vector.
Thus, a loxP/FRT/PGK/Tn5-neo-pA/FRT cassette is flanked by the homology arms in
Foxc2 3’-loxP inserting vector. The Foxc2 3’-loxP inserting vector was linearized by
I-SceI and electropolated into the SW102 carrying Foxc2 ‘5-loxP vector in which
recombineering proteins were induced by heat (42oC) treatment (Step VII). Successfully
recombineered colonies carrying resultant Foxc2 loxP-neo vector were selected by
kanamycin and ampicillin. To confer zeosin resistance to the Foxc2 loxP-neo vector, a
1.3 kb fragment of SV40/EM7-Sh ble-pA cassette flanked by rox sequence was amplified
by two step PCR using KOD plus (Toyobo) and pSV40/Zeo2 (Invitrogen, Carlsbad, CA)
as a template. First PCR amplified the SV40/EM7-Sh ble-pA cassette and add rox
sequences to each end by using primers ‘k’ and ‘l’. Second PCR was designed to add
homology arms to the PCR product of first PCR. Thus, 5’- upstream loxP sequence (64
bp) was added to the forward primer ‘m’ and Foxc2 genome sequence (55 bp) just
downstream to the loxP site was added to the reverse primer ‘n’ (Step VIII). Finally, the
PCR product, i.e. loxP/rox/SV40/EM7-Sh ble-pA/rox/Foxc2 genome fragment, was
electroporated into the heat-treated SW102 carrying the Foxc2 loxP-neo vector (Step IX).
Positive clones carrying resultant conditional Foxc2 targeting vector were selected by
zeosin and ampicillin.
Computed tomography (CT) scan of newborn mice
CT scan of newborn mice were performed according to the method described by
Tamura et al (S1). Briefly, newborn mice were sacrificed by overdose of sodium
pentobarbital and their skins were removed. Whole bodies were fixed overnight in 10%
buffered neutral formalin solution, and washed twice with PBS. The fixed mice were
then immersed in contrast media containing 9 mM iodine (Nacalai Tesque, Kyoto,
Japan) and 15 mM potassium iodide (Nacalai Tesque) for 20 hours. The whole bodies
were scanned along the body axis at 48 µm intervals by an X-ray CT scanning device for
experimental animals (Latheta LCT-200, Hitachi Aloka Medical, Tokyo, Japan)
according to manufacturer’s instruction. The images were analyzed by 3D slicer
(http://www.slicer.org/).
Supplementary reference
S1. Tamura M, Hosoya M, Fujita M, Iida T, Amano T, Maeno A, Kataoka T, Otsuka T,
Tanaka S, Tomizawa S, Shiroishi T. (2013) Overdosage of Hand2 causes limb and heart
defects in the human chromosomal disorder partial trisomy distal 4q. Hum. Mol. Genet.
22 (12): 2471-2481 doi:10.1093/hmg/ddt099.
Supplementary figure legends
Figure S1. Schematic presentation of the construction of Foxc2 targeting vector
Figure S2. Computed tomography (CT) scan of newborn mice
CT images from a hetero-floxed mouse (left panels) and a homo-floxed mouse (right
panels) are shown. Although the thoracic cavity spaces were almost the same, the
pleural space, i.e. the space between the rib cage and lungs were more prominent in the
homo-floxed mouse than that in the hetero-floxed mouse, indicating that lung expansion
after birth was insufficient.
Supplemental table S1.
Primer usage
Primer sequence
TGCTCCACAGAAGCAAGGAGGCCAGGTCCTGCGCGAA
a
Insert 5'-loxP sense
GGAACGAGCCTCCCCGGTACCAAACCCTATGCTACTCC
GTC
b
Insert 5'-loxP antisense
CTCAAATGGATCGACCCACGACCCCTCTCCTAAAGAAA
GAGTGGGGGCTCCTATTTGTCCTACTCAGGAGAGCG
Retrieve from the BAC
c
clone 5'-upstream
GGCCTCGGAGGCCGACTTGTGAGTCACCGCATAG
homology arm sense
Retrieve from the BAC
d
clone 5'-upstream
GGCCAACCCGGCCTGCTTGGCTGGACAGGTT
homology arm antisense
Retrieve from the BAC
e
clone 3'-downstream
GGCCACTTAGGCCTGTGGGTTTATCCCAAGTACAG
homology arm sense
Retrieve from the BAC
f
clone 3'-downstream
GGCCCTTACGGCCGGTTTGTGCTTTGAGGTGTAG
homology arm antisense
Insert 3'-loxP
g
5'-upstream homology
GGCCTCGGAGGCCCCATGGGAACCTTCTTCGAC
arm sense
Insert 3'-loxP
h
5'-upstream homology
GGCCAACCCGGCCCACCTTATCCGGAGAGACCT
arm antisense
Insert 3'-loxP
i
3'-downstream homology
GGCCACTTAGGCCCCTTCTGTAAACGAGTGCGG
arm sense
Insert 3'-loxP
j
3'-downstream homology
GGCCCTTACGGCCACTGTACAAAGCCATGCACTTC
arm antisense
k
Amplify Sh ble expression GCGGTCTCTCCTGTAACTTTAAATAATTGGCATTATTTA
cassette sense
AAGTTAGAGGGTGTGGAAAGTCCCCA
l
m
n
Amplify Sh ble expression TTAACTTTAAATAATGCCAATTATTTAAAGTTACAGACA
cassette antisense
Insert Sh ble expression
cassette sense
TGATAAGATACATTGATGAGTTTGG
GGTACCAAACCCTATGCTACTCCGTCATAACTTCGTAT
AGCATACATTATACGAAGTTATCCTGTAACTTTAAATAA
TTGGC
Insert Sh ble expression
CTCAAATGGATCGACCCACGACCCCTCTCCTAAAGAAA
cassette antisense
GAGTGGGGGCTCCTGCTTAACTTTAAATAATGCCAATT
Underline in a: KpnI site, Underline in c-j: SfiI site.
Figure S2.