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Developmental Biology

Effektiv Generering av hiPSC Neural Lineage Specifika Knockin Reportrar Använda crispr / Cas9 och Cas9 Dubbel Nickase System

doi: 10.3791/52539 Published: May 28, 2015
*1,2, *1,2, 1,2,5, 1,2,6, 1,2,4,7, 1,2,3
* These authors contributed equally

Abstract

Riktad genmodifiering är ett kritiskt förhållningssätt för att karakterisera genfunktioner i modern biomedicinsk forskning. Emellertid har effektiviteten hos genmålsökning i humana celler varit låg, vilket förhindrar alstring av humana cellinjer med en önskad hastighet. De senaste två åren har det skett en snabb progression på att förbättra effektiviteten i genetisk manipulation av genomet redigeringsverktyg såsom crispr (Clustered regelbundet mellanrum Kort palindrom upprepar) / Cas (crispr-associerade) systemet. Detta manuskript beskriver ett protokoll för att generera härstamning specifika mänskliga inducerade pluripotenta stamceller (hiPSC) reportrar använder crispr / Cas systemet assisterad homolog rekombination. Rutiner för att erhålla nödvändiga komponenter för att göra neurala härstamning reporter linjer använder crispr / Cas-system, med fokus på konstruktion av målstyrningsvektorer och enstaka styr RNA, beskrivs. Detta protokoll kan utvidgas till plattform etablering och mutation korrigering hiPSCs.

Materials

Name Company Catalog Number Comments
pStart-K Addgene 20346
pWS-TK6  Addgene 20350
pKD3  Addgene 45604
pKD46 The Coli Genetic Stock Center, CGSC 7634
PGK-neo-bpA sequence  Addgene 13442
Human BAC clones of target genes  https://bacpac.chori.org
JDS246 (Cas9-003), Mammalian codon-optimized streptococcus pyogenes Cas9-3X Flag Addgene 43861
MLM3636, Human-gRNA-ExpressionVector with U6 promoter  Addgene 43860
pX335-U6-Chimeric_BB-CBh-hSpCas9n(D10A) Addgene 42335
sgRNA design, ZiFit http://zifit.partners.org/ZiFiT/
Off target prediction tool CasOT http://eendb.zfgenetics.org/casot/download.php
Perl http://www.perl.org/get.html
NCBI http://www.ncbi.nlm.nih.gov/
BLAT https://genome.ucsc.edu/cgi-bin/hgBlat?command=start
sgRNA Primer synthesis Sigma
One shot Top10 Electrocomp E. Coli Competent cells Life Technologies C4040-50
AccuPrime Pfx SupperMix Life Technologies 12344-040
Restriction enzymes NEB and Life Technologies
Zymoclean Gel DNA Recovery Kit Zymo Researech D4007
DNA quick extraction buffer  Epicentre QE0905T
Herculase II Fusion DNA Polymerases Agilent Technologies 600675
Digestion buffer 2 New England Biolab
6X DNA Loading Dye Thermo Scientific R0611
TeSR-E8 Kit for hESC/hiPSC Maintenance Stem Cell Technologies 05940
UltraPure Agarose  Life Technologies 16500-100
50xTAE Life Technologies B49
Essential 8 medium  Life Technologies A1517001
Dulbecco’s Phosphate Buffered Saline without Calcium and Magnesium  Life Technologies A12856-01
D-MEM/F12 with Glutamax  Life Technologies 10565018
D-MEM with Glutamax  Life Technologies 10566040
Fetal Bovine Serum-ES cell qualified  Life Technologies 10439
Knockout serum replacement  Life Technologies 10828010
2-mercaptoethanol 1000X  Life Technologies 21985023
Non Essential Amino Acid  Life Technologies 11140050
Stempro Accutase  Life Technologies A1110501
Dispase  Life Technologies 17105-041
0.25% Trypsin- EDTA solution  Life Technologies 25200-056
Geltrex  Life Technologies    12760-013
ROCK inhibitor Y-27632 Millipore    SCM075
SMC4 reagent  BD 354357
Neomycin resistant MEF  Millipore PMEF-NL
Hygromycin resistant MEF  Millipore PMEF-HL
G418 (Geneticin) LifeTechnologies 11811
Hygromycin B  Life Technologies 10687010
FIAU (Fialuridine, 1-2-Deoxy-2-fluoro-ß-D-arabinofuranosyl-5-iodouracil  Moravek Biochemicals and Radiochemicals    M251
Electroporator: Gene Pulser Xcell  Bio-Rad
0.4 cm electroporation cuvette  Bio-Rad 165-2088
DIG-High prime DNA labeling and detection starter kit II  Roche 11585614910
Hybridization denature solution  VWR 82021-478
PCR DIG probe synthesis kit  Roche 11636090910
DIG wash set  Roche 11585762001
Anti-Digoxigenin (DIG-AP) Roche 11093274910
CSPD chemiluminescence system  Roche 11755633001
DIG wash and block buffer set  Roche 11585762001
50X TAE buffer  Life Technologies 24710030
Blotting buffer (25 mM Tris pH 7.4,  0.15 M NaCl,  0.1% Tween20)
Hoefer Ultraviolet Crosslinker  Fisher Scientific 03-500-308
Spermidine  Fisher AC13274-0010
Tris HCl 2M (pH 7.5) VWR    200064-506
Denville Scientific blue bio film 8x10  Fisher nc9550782
DNA molecular weight marker II ( DIG-labeled ) Roche 11218590910
Amersham Blotting membrane Hybond-N+  Roche 95038-400
Pyrex glass drying tray  Fisher 15-242A
Kimberly-Clark C-fold paper towels  Fisher 06-666-32B
Whatman 3MM paper (26X41) Fisher    05-713-336
Hybridization bag  Roche 11666649001
Hybridization tubes  Fisher 13-247-300
Hybridization oven rotisserie Shake 'n' Stack  Fisher HBMSOV14110

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References

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Effektiv Generering av hiPSC Neural Lineage Specifika Knockin Reportrar Använda crispr / Cas9 och Cas9 Dubbel Nickase System
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Cite this Article

Li, S., Xue, H., Long, B., Sun, L., Truong, T., Liu, Y. Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System. J. Vis. Exp. (99), e52539, doi:10.3791/52539 (2015).More

Li, S., Xue, H., Long, B., Sun, L., Truong, T., Liu, Y. Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System. J. Vis. Exp. (99), e52539, doi:10.3791/52539 (2015).

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