Adachi413Supl - BioTechniques

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Adachi413Supl - BioTechniques
Short Technical Reports
Supplementary Material For:
Simple one-week method to construct gene-targeting vectors:
application to production of human knockout cell lines
Susumu Iiizumi1, Yuji Nomura1, Sairei So1, Koichi Uegaki1, Kayoko Aoki1, Kei-ichi Shibahara2, Noritaka Adachi1, and
Hideki Koyama1
1Yokohama
City University, Yokohama and 2National Institute of Genetics, Mishima, Japan
Iiizumi et al. Supplemental Fig
BioTechniques 41:XXX-XXX (September 2006)
XhoI
XhoI
NdeI
att R3
ccd B
att R4
pMC1
pDEST R4-R3
4.1 kb
Invitrogen
Cat No. :12537-023
r
Amp
r
Amp
NdeI digestion
Subcloning
DT-A
PGK-pA
pMC1 DT-A
4.33 kb
KURABO
Cat No. : MK-1003
XhoI digestion
AatII
att R4
ccd B
att R3
PGK-pA
DT-A
MLS fragment
1
pMC1
r
20
30
40
50
������������������������������������������������������ 3'
5'
3' ������������������������������������������������������
5'
pDEST R4-R3 DT-A
Amp
10
PmeI
5.4 kb
AscI
I-SceI
SwaI
PacI
(NdeI/XhoI sites are deleted)
Aat II digestion
Insertion
PacI
SwaI
I -SceI
att R4
ccd B
att R3
PGK-pA
DT-A
pMC1
AscI
PmeI
pDEST DTA-MLS
r
Amp
5.4 kb
(AatII site is deleted)
Supplementary Figure S1. pDEST DTA-MLS. DT-A, a gene that codes for a diphtheria toxin A fragment; MLS, multiple-linearization site.
Vol. 41 ı No. 3 ı 2006
www.biotechniques.com ı BioTechniques ı 1
Short Technical Reports
A
A
att B1 primer
att B2 primer
at
t
B
HindIII BamHI
BamHI
HindIII
Hyg r
PolyA
Hyg r
PolyA
pPGK
pPGK
att L1
NotI
His r
NotI
Hyg r
PolyA
pPGK
att L1
yA
Km
Hyg r
pPGK
r
att L2
pβActin
PolyA
att L1
His r
pβActin
NotI
His r
PolyA
pβActin
Puro r
pPGK
Sal I
PolyA
Subcloning
NotI
NotI
pPGK
pBS lox-His
6.2 kb
Puro r
NotI
pPGK
PolyA
p
pBS lox-Puro
4.2 kb
NotI digestion
Subcloning into pENTR loxP
NotI digestion
Subcloning into pENTR loxP
EcoRV
pBS246
pPGK Puro 2.5 kb
4.9 kb
EcoRV digestion
SalI digestion
Subcloning
pβActin
D
BamHI
D
EcoRV
att L1
att L2
PolyA
PolyA
r
Km
pβActin
His r
att L2
PolyA
PolyA
pPGK
att L1
pENTR lox-His
6.6 kb
r
Km
NotI digestion
Subcloning into pENTR loxP
Puro r
att L1
att L2
PolyA
pENTR lox-Puro
4.6 kb
Puro r
pPGK
pBS246
2.5 kb
PolyA
pPGK Puro
4.9 kb
EcoRV digestion
SalI digestion
Subcloning
NotI
NotI
ctin
NotI
pPGK
Puro r
PolyA
pBS lox-Puro
4.2 kb
NotI digestion
Subcloning into pENTR loxP
digestion
oning into pENTR loxP
att L2
att L1
s
r
pPGK
Puro r
PolyA
att L2
pENTR lox-Puro
4.6 kb
Km
Supplementary Figure S2. Entry clones for positive selectable markers. (A) pENTR loxP; (B) pENTR lox-Hyg; (C) pENTR lox-His; and (D) pENTR loxPuro. Hygr, hygromycin-resistance gene; Hisr, histidinol-resistance gene; Puror, puromycin-resistance gene.
2 ı BioTechniques ı www.biotechniques.com
pPGK
pEN
r
Km
Sal I
SalI
pβActin
pβActin His
6.4 kb
His r
pENTR lox-His
6.6 kb
r
Km
bcloning
olyA
olyA
BamHI
SalI
pβActin
pBS246His
6.4 kb
kb
2.5
NotI
pBS lox-His
6.2 kb
pENTR lox-Hyg
4.9 kb
pENTR lox-Hyg
r
4.9 kb
Km
His r
D
att L2
pENTR loxP
att L1
2.9 kb
att L2
PolyA
Hyg r
pPGK
His r
PolyA
r
BamHI digestion EcoRV digestion
NotI
NotI digestion
Subcloning into pENTR loxP
NotI
NotI
NotI digestion
Subcloning into pENTR loxP
BP recombination
pPGK
PolyA
Subcloning
pBS lox-Hyg
4.6 kb
pBS lox-Hyg
4.6 kb
att L1
Hyg r
PolyA
Km
EcoRV
pβActin
BamHI digestion
NotI
NotI
att L2
pENTR loxP
2.9 kb
BamHI
BamHI
BamHI
pBS246
pβActin His
2.5 kb
6.4 kb
Subcloning
Subcloning
att L1
att L2
D
Iiizumi et al. Supplemental Figure 2
NotI
att P2
C
C
PolyA
pPGK-Hyg
pBS246
4.9 kb
2.5 kb
HindIII, BamHI digestion
HindIII, BamHI digestion
r
BamHI
BamHI
BamHI
ccd B
NotI
NotI
NotI
BP recombination
NotI
pDONR 221
4.8 kb pENTR loxP
2.9 kb
Invitrogen
r No. : 12537-023
Cat.
Km
Km
C
HindIII
att B2
att P1
att P2
pDONR 221
4.8 kb
Invitrogen
Cat. No. : 12537-023
pBS246
pPGK-Hyg 2.5 kb
4.9 kb
pBS246
2.5 kb
ccd B
att P1
r
B
PCR
pBS246
att B1
2.5 kbatt B2
Gibco
BP recombination
Cat.No. : 10348-019
att B1
Km
HindIII BamHI
B2
B1
B1
B2
t
at
PCR
pBS246
2.5 kb
Gibco
Cat.No. : 10348-019
B
att B2 primer
at
t
att B1 primer
t
at
A
Iiizumi et al.Figure
Supp
Iiizumi et al. Supplemental
Vol. 41 ı No. 3 ı 2006

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