TALEN

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TALEN
TALEN
TALEN
TALEN


ID
NCBI
1.1

Search

1.2 homo sapiens

1.3

GeneBank

1.4 CDS

1.5 online TALEN designhttps://talent.cac.cornell.edu/DNA Star

SeqbuilderFileNew DNA

1.6 CDS

TALEN
TALEN
TALEN
TALEN

TALEN

112-19bp
2spacer1221bp
3 TT
4 spacerGC

TALEN
2X3TALEN

Spacer 12-21bp

TALEN
TALEN
TALEN
TALEN

Day1: TALEN

PCR4

Day2:TALEN

5ml Ka+LB

Day3:4-5ml

plasmid
Buffer G
ddH2O
BamH I
Pst I

10 ul
2 ul

1 Unit
1.5 Unit
20ul

4526bp

2061bp

305
306

Day4:NCBI BLAST

blast

Day4:TALEN

Blast100%

TALEN
TALEN
TALEN
TALEN


1TALEN
TALEN
2

300ng/ul
OD260/280=1.8

TALEN
2ug
EGFP
0.5ug
VOpti-MEMV = 50ul1ug
VFugeneV = 3ul1ug

DNA

PCR
PCR
DNAPCR

Marker

560bp

PCR

---
A

BPCRT

C
30-50

TALEN

TALEN

Genomic PCR

Zebrafish-gene A

DNA
580bp

PCR

Restrictive enzyme digest

TALENspacer

Zebrafish-geneA (Kpn I)

580bp
401bp
179bp

80%

 T7E1assay
DNADNA Holliday
DNADNA

72hDNA

PCR
PCR9450-60
T7E1371h

NATURE BIOTECHNOLOGY ,2013, 31(3), 251-8

TALEN
TALEN
TALEN
TALEN

/
TALEN
Donor

TALEN

puromycin

mix,DNA
PCRTA
30-50

96

,
30%100

100*30%
*30% *2/3*2/3=6

,
30%100

100*30%*2/3=20

TALEN

Puromycin

mix,DNA
PCRTA
30-50

96

Knock-in
knock-in

WT

Donor

Su Mi Choi, Yonghak Kim,efficient drug screening and gene correction for treating liver disease using
patient-specific stem cells . HEP,12-2140.

Dirk Hockemeyer, Haoyi Wang, Samira Kiani.Genetic engineering of human ES and iPS cells using TALE
nucleases,Nat Biotechnol. ; 29(8): 731734.

TALENKO
----
HelaXX

TALEN
TALEN

100%


Hela
puromycin3

DNA, PCR

PCR

TALEN

PCRTA

53%100
53

96

50

2014/5/21


knock-out
293Thela100%
1930 % -70%
2

ES/iPS100%
720 % -50%
4
2013.07


knock-in
293Thela100%
620 % 55%3

ES/iPS100%
25.5
10 % -28%
2013.07

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