Abstract
استهداف الجين هو مقاربة نقدية لوصف وظائف الجينات في البحوث الطبية الحيوية الحديثة. ومع ذلك، فإن فعالية استهداف الجينات في الخلايا البشرية المنخفضة، مما يمنع جيل من خطوط الخلايا البشرية بمعدل المطلوب. وقد شهدت خلال العامين الماضيين تطورا سريعا على تحسين كفاءة التلاعب الجيني بواسطة أدوات تحرير الجينوم مثل كريسبر (المجمعة بانتظام Interspaced قصيرة المتناوب يكرر) / كاس (المرتبط كريسبر) النظام. تصف هذه المخطوطة بروتوكول لتوليد محدد الناجمة عن النشاط البشري الخلايا الجذعية المحفزة (hiPSC) للصحفيين النسب باستخدام النظام كريسبر / كاس بمساعدة إعادة التركيب مثلي. إجراءات الحصول على المكونات اللازمة لصنع خطوط مراسل النسب العصبية باستخدام كريسبر / نظام CAS، مع التركيز على بناء استهداف ناقلات والرنا دليل واحد، ويرد. ويمكن تمديد هذا البروتوكول لإنشاء منصة وتصحيح طفرة في 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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