TALENknock-out /knock-in
TALEN/
SIDANSAI Biotechnology CO.,LTDTALENTALENTALENTALEN
IDNCBI
1.1 Search
1.2 homo sapiens
1.3 GeneBank
1.4 CDS
1.5 online TALEN designhttps://tale-nt.cac.cornell.edu/DNA Star
SeqbuilderFileNew DNA
1.6 CDS
TALENTALENTALENTALEN
TALEN112-19bp2spacer12- 21bp3 TT4 spacerGC
TALEN
Spacer 12-21bp5533 2X3TALENTALENTALENTALENTALEN
Day1: TALEN
PCR4
Day2:TALEN
5ml Ka+LB4
Day3:4-5ml plasmid 10 ul Buffer G 2 ul ddH2O BamH I 1 Unit Pst I
1.5 Unit 20ul
305306
blast
Day4:NCBI BLAST
Blast100% Day4:TALEN
TALENTALENTALENTALEN
1TALENTALEN 2300ng/ulOD260/280=1.8
DNATALEN 2ugEGFP 0.5ug VOpti-MEMV = 50ul1ugVFugeneV = 3ul1ug
PCRPCR
MarkerPCRDNAPCR560bp
---
A
BPCRT
C 30-50
TALEN
TALENDNAPCR
Zebrafish-gene AWT580bpZebrafish-geneA (Kpn
I)WTTALEN580bp401bp179bpGenomic PCRRestrictive enzyme digest
TALENspacer80%
25 T7E1assayDNADNA Holliday DNADNA
72hDNAPCRPCR9450-60T7E1371h
NATURE BIOTECHNOLOGY ,2013, 31(3), 251-8
TALENTALENTALENTALEN
TALENpuromycin mix,DNAPCRTA30-50+
TALENDonor/96,30%100100*30%*2/3=20,30%100100*30% *30%
*2/3*2/3=6TALENPuromycinmix,DNAPCRTA30-5096Knock-in knock-in
WTDonor
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----HelaXXTALENTALEN
100%
34Helapuromycin3DNA, PCRPCR
TALEN
53%10053PCRTA
2013-8-22396
350
knock-out293Thela100%1930 % -70%2
ES/iPS100%720 % -50%4
2013.07
knock-in 293Thela100%620 % -55%3
ES/iPS100%25.510 % -28%
2013.07
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