Genome-TALER™ Human AAVS1 Safe Harbor Gene Targeting Kit
Contents
1. identify clones with correct insert 4 Inoculate a positive colony containing insert in an appropriate amount of LB Ampicillin Carbenicillin Broth Incubate at 37 C overnight Extract and purify the construct using an endotoxin free plasmid purification kit Sequence verification of the insert is optional Co transfection of AAVS1 TALEN pair and donor plasmid 1 Plate 100 000 to 300 000 cells well in a 6 well plate following the recommended conditions for cell type s being transfected Include wells for the following On the day before transfection trypsinize and count the cells The number of cells plated in each well should be determined so that they are 70 80 confluent at the time of transfection a AAVS1 TALENS positive control DC RFP SH01 b Positive control DC RFP SHO1 only c AAVS1 TALENSs donor in vector DC DON SHO1 d Donor in vector DC DON SHO1 only 2 Next day prepare transfection complexes of AAVS1 TALENs pair plasmids and donor plasmids using suitable transfection reagents according to the manufacturer s instructions Leave the transfection complexes on the cells to react for 6 hours Example For HEK293T cells using 6 EndoFectin Plus Transfection Reagent transfect 0 5ug of each TN AAVS1 L and TN AAVS1 R vectors 1ug total and 1ug of donor vector Tech Notes 1 Since transfection efficiencies vary across different cell lines we recommend optimizing the input of AAVS1 TALENS pair plasmids to donor vectors f2. 500 ng ul DC DON SH01 AAVS1 donor vector 10 ug 500 ng ul DC RFP SHO1 AAVS1 RFP control 10 ug 500 ng ul HQPAVSHR 5 5 HR primer pair 200 reactions 10 uM HQPAVSHR 3 3 HR primer pair 200 reactions 10 uM Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit A TALEN and donor plasmids AAVS1 R AAVS1 Right TALEN AAVS1 Left TALEN bGH poly A bGH poly A bGH poly A Puro E 4 3 i AAVS1 o E Donor control i a J hee HA Right Core Insulator AAVS1 HA Right B Knock in verification PCR primers 5 F primer 3 F primer ida ida Chr 19 GOI bGHpA EFla GFP T2A Puro SV40pA HAR lt 5 R primer 3 R primer cs 1 2kb Figure 2 Safe harbor gene targeting kit components A TALEN and donor plasmids B Knock in verification primer pairs Acknowledgement Design of the AAVS1 left TALEN AAVS1 right TALEN and AAVS1 donor control vectors was performed by Dr Jizhong Zou of the NIH Center for Regenerative Medicine a Common Fund initiative of the U S National Institutes of Health Additional materials required LB Agar and broth containing 50 ug ml kanamycin 6 well tissue culture plates and related tissue culture supplies Other specific media and additives specific for cell type of interest Any high transformation efficiency RecA and EndA E coli competent cells Dulbecco s Modified Eagle s Medium D MEM high glucose with sodium pyruvate and glutamine Invitrogen Cat 11995073 6
3. EndoFectin Plus Transfection Reagent Genecopoeia Cat EFP1003 01 02 7 Qiagen EndoFree Plasmid Maxi Kit Qiagen Cat 12362 8 Qiagen DNeasy Blood and Tissue Kit Qiagen Cat 69504 9 iProof High Fidelity DNA Polymerase BioRad Cat 172 5301 10 Fetal Bovine Serum Invitrogen Cat 16000036 11 Penicillin Streptomycin Invitrogen Cat 15070063 12 Trypsin EDTA Sigma Cat T3924 13 Optional For difficult to transfect cells the use of an electroporation system e g Lonza s NucleoFector or Invitrogen s Neon system is highly recommended So Sv40 poly A Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit Ill Example bGH poly A AAVS1 Donor control AAVST HA Left Sv40 poly A amp Core Insulator AAVS1HA Right dad Rr AAVS1 AAVS1 Left TALEN Right TALEN C oe 5 F primer 3 F primer a Chr 19 GOl bGHpA EFla 3FP T2A Puro SV40pA HAR lt 5 R primer 3 R primer p s 1 2kb Figure 3 Human genome safe harbor D AAVS1 gene targeting b M 1 2 3 4 A AAVS1 donor control plasmid P pAAVS1D RFP copGFPpuro 800 ng was 3000 2500 co transfected with AAVS1 TALEN pair 600 2000 ng for each or control TALEN pair into HEK293T cells in a 6 well pate B 48 hr post transfection the cells were split 1 10 into a new 6 well pate 1500 1250 de ue leet 1000 T90 and selected against 1 0 ug ml o
4. GeneCopoeia Expressway to Discovery Genome TALER Human AAVS1 Safe Harbor Gene Targeting Kit Catalog SH AVS K100 User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry 2genecopoeia com WWW genecopoeia com 2013 GeneCopoeia Inc Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit USER MANUAL Genome TALER Human AAVS1 Safe Harbor Gene Targeting Kit l Introduction ll Content and Storage Il Example IV Procedure V Reference Vl Licensing and Warranty Statement I Introduction Background of TALEN Transcription activator like TAL effectors can recognize and bind host plant promoter sequences through a central repeat domain consisting of a variable number of 34 amino acid repeats The residues at the 12th and 13th positions of each repeat appears to be a simple one to one code to each DNA base of target sequence e g NI A HD C NG T and NN GorA TAL effectors have been utilized to create site specific gene editing tools by fusing target sequence specific TAL effectors to nucleases TALENs transcription factors TALE TFs and other functional domains These fusion proteins can recognize and bind chromosome target sequences specifically and execute their gene editing functions such as gene knockout knockin with donor plasmid modification activation repression and more Safe gene targeting The mod
5. dation of either the 5 or 3 homology arms for donor integration is usually sufficient however both arms can be done for additional confirmation Primer name HQPAVSHR 5F Primer sequence See datasheet HQPAVSHR 5R See datasheet HQPAVSHR 3F See datasheet See datasheet 2 Protocol details for junction PCR assay a Isolate genomic DNA from positive control cells or test sample cells using a Suitable genomic DNA miniprep kit Please follow protocol recommended by the manufacturer b Perform junction PCR PCR reaction below TALEN cut positive Positive control control donor donor only Genomic DNA 60 100ng uol 1 ul 1 ul 5uM 5 AAVS1 PCR Primer Mix 1 ul 1 ul 5XRD Buffer Sul 5ul 5XEnhancer Sul Sul Reagent 25mM dNTPs 20mM MgSO4 FL265 2mg ml UltraPF 5U ul PCR grade distilled water Total 0 2ul 2 5ul 0 275ul 0 25ul 11 775yl 25yul 0 2ul 2 5ul 0 275ul 0 25ul 11 775yl 25yul Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit 98 C 5min 98 C 20sec 55 C 30sec 35 cycles 72 C 1min 72 C 7min Hold at 4 16 C Run the PCR reaction out on the 1 Agarose EtBr gel in 1X TAE buffer to confirm the Junction PCR result Sample results for 5 and 3 Junction PCR Assay shown below 5 F primer 3 F primer Chr 19 GOI bGHpA EF la GFP T2A Puro SV40pA HAR lt lt 5 R primer 3 R primer a 1 2kb V Reference 1 Zou J et al 2009 Gene targeting of a d
6. ed pDonor SH01 empty vector 7 0 ul DNA insert 30 50 ng or water control 3 Incubate reactions at 25 C for 1 2 hours sticky end ligation or O N at 16 C for blunt end ligation 2 Transformation Transform competent cells transformation efficiency at least 1x10 colonies ug pUC19 with the whole ligation reaction 10ul following the provided protocol of the competent cells Plate the transformed competent cells on LB Ampicillin Carbencillin agar plates 3 Screening correct clones 1 Depending on the ratio of colony numbers for the cDNA sample vs the negative control sample randomly mark 5 or more well isolated colonies 2 Prepare a PCR Master Mix with PCR primers flanking the insert trxn 10rxn Composition 2 5ul 25ul 10X PCR Reaction Buffer 21 9yul 219yl Nuclease free water O 2ul 2ul Taq DNA polymerase approx 5 U ul 25ul 250yl Total volume 3 Mix the master mix very well and aliquot 24ul into each well of 96 well PCR plate or individual tubes 4 Pick the each marked colony from step 1 using sterilized tips and mix it to each well or tube Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit 5 Proceed with PCR using the following program 94 C 4 min 1 cycle 94 C 0 5 min then 68 C 1 min 1 kb 25 cycles 68 C 3 min 1 cycle Depending on the size of final PCR product use a shorter or longer time 6 Take 5ul of the PCR reaction and run it on a 1 2 agarose EtBr gel in 1X TAE buffer to
7. f puromycin The images were taken after two weeks of selection The cells transfected with pAAVS1D RFP copGFPpuro control TALEN were completely killed by puromycin selection data not shown C D PCR primers designed to amplify the HR junction were used to verify the successful integration Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit IV Procedure Plasmid propagation We recommend propagating the plasmids provided in the safe harbor kit before the gene targeting experiment Plasmids can be transformed using standard conditions suitable in any RecA and EndA E coli competent cell For transformation of AAVS1 TALENs we suggest plating 50 200ul of transformed cells on fresh LB Kanamycin plates 50ug ml For plasmids in pDonor SHO1 vector use LB Ampicillin plates 50ug ml Incubate the plates at 37 C overnight Inoculate colonies from the transformation and grow them at 37 C overnight in 200ml of LB media containing either 50ug ml of Kanamycin AAVS1 TALENSs or Ampicillin pDonor SHO1 vector Use an endotoxin free plasmid DNA maxiprep kit to extract plasmid DNA after the overnight growth To confirm integrity of the amplified plasmids we recommend restriction digestion analysis or direct sequencing Cloning into empty pDonor SHO1 vector 1 Ligation 1 Digest and gel purify the vector plasmid Dilute it to 10ng ul 2 Setup 10ul ligation reaction for each control and test sample Volume Item 1 0 ul Digest
8. fe integration Designated AAVS1 human genome safe harbor integration site ensures transcription competency of the transgenes and presents no known adverse effect Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit Specific targeting TALEN mediated DNA DSBs at the AAVS1 site stimulate homologous recombination dramatically for transgene integration Known copy number Known copy number of the transgene ensures predictable expression level and simplifies phenotype interpretation Compatible knock in ORFs Over 18 000 sequence verified human ORFs are compatible for transgene donor DNA design Chr 14 MS lacus TALEN paar ij Dong Clone a i im damas Target gene di kn ck in AWS 1 opus GD Dr ED LU ELE Figure 1 Illustration of TALEN mediated transgene integration at the safe harbor AAVS1 site of human genome ll Content and storage Genome TALER human AAVS1 safe harbor gene targeting kit Cat SH AVS K100 Shipped at room temperature Stored at 20 C Shipped at room temperature Stored at 20 C Shipped at room temperature Stored at 20 C Shipped at room temperature Stored at 20 C Shipped at room temperature Stored at 20 C Shipped at room temperature Stored at 20 C AAVS1 donor clones can be custom made and included in the kit instead of the empty AAVS1 donor cloning vector TN AAVS1 L AAVS1 left TALEN 10 ug 500 ng ul TN AAVS1 R AAVS1 right TALEN 10 ug
9. ification of the human genome by insertion of genes of interest and other genetic elements in unique site s of chromosome s is of great value for cell engineering The genetically modified cells are valuable for therapeutic research gene function study as well as lineage tracking and analysis All these applications depend on the reliable and predictable function of the transgene without perturbing any endogenous gene and or other regulation element Random integration of the transgene on the contrary can present a threat of unpredicted insertion or mutagenesis The new approach recently developed is to deliver the transgene to a predetermined and safe site in a genome AAVS1 also known as PPP1R2C locus in human chromosome 19 is a well validated safe harbor to host the DNA fragment with expected function It has an open chromatin structure and is transcription competent Most importantly there is no known adverse effect on the cell resulting from the inserted DNA fragment of interest Genome TALER human AAVS1 safe harbor gene targeting kit is designed to specifically transfer your gene of interest selection marker or other genetic element from a donor plasmid into the AAVS1 safe harbor site in human chromosome 19 via TALEN mediated homologous recombination HR HR is a natural DNA repair mechanism that occurs in response to DNA double strand breaks DSBs The DSBs here are created by AAVS1 specific TALENS figure 1 Advantages Sa
10. isease related gene in human induced pluripotent stem and embryonic stem cells Cell Stem Cell 2009 Jul 2 5 1 97 110 2 Sadelain M et al 2011 Safe harbours for the integration of new DNA in the human genome Nat Rev Cancer 2011 Dec 1 12 1 51 8 3 van Rensburg H et al 2013 Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hepatopoietic stem cells Gene Therapy 2013 20 2 201 14 4 Papapetrou EP et al 2011 Genomic safe harbors permit high B globin transgene expression in thalassemia induced pluripotent stem cells Nat Biotechnol 2011 29 1 73 8 5 Lombardo A et al 2011 Site specific integration and tailoring of cassette design for sustainable gene transfer Nat Methods 2011 8 10 861 9 VI Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of the Genome TALER Human AAVS1 Safe Harbor Gene Targeting Kit the Product If the terms and conditions are not acceptable the Product in its entirety must be returned to GeneCopoeia within 5 calendar days A limited End User license is granted to the purchaser of the Product The Product shall be used by the purchaser for internal research purposes only The Product is expressly not designed intended or warranted for use in humans or for therapeutic or diagnostic use The Product must not be resold repackaged or modified for resale or used to manufacture commercial prod
11. or best results We recommend starting with a 1 1 ratio e g 1ug of donor HR plasmid and 0 5ug of each TALE Nuclease plasmid 2 For optimal results we recommend complexing DNA with transfection reagent in serum and antibiotic free media and cells growing in complete media e g DMEM F12 10 FBS w o antibiotics 3 For hard to transfect cells e g primary stem hematopoietic it may be advisable to utilize a non passive transfection method Please follow recommended guidelines provided by the manufacturer for the specific cell type s being transfected 3 24 hours post transfection remove transfection media and split the cells 1 10 and 1 20 in complete growth media w antibiotics Plate cells into 6 well plates and save a set of plate s for characterization of samples by junction PCR assay see below Allow cells to recover for 24 hours 4 Begin puromycin selection 48 hours post transfection For 293T cells the recommended concentration of puromycin is 1 ug ml Tech Note Establishing a kill curve on untransfected cells can determine the effective working puromycin concentration for a target cell line The concentration of puromycin typical working range of 0 5ug 5ug ml that kills 29096 of cells after 48hours of selection is the correct dose for the cells being selected Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit Validation of TALEN modified and HDR recombinant cells 1 Assay for TALEN cutting and HDR of dono
12. r vectors on samples as follows 1 AAVS1 TALENS positive control DC RFP SH01 Select cells in Puromycin for 7 10 days resulting colonies should be RFP amp GFP positive 2 Positive control DC RFP SHO1 only Select cells in Puromycin for 7 10 days very few colonies if any should be seen comparing to positive control donor AAVS1 TALENs Sample a Presence of PuroR RFP GFP colonies indicates frequency of random integration events 3 AAVS1 TALENs donor in vector DC DON SHO1 Select cells in Puromycin for 7 10 days colonies should be GFP positive Expression of insert may be detected by qPCR or Western blot 4 Donor in vector DC DON SHO1 ony Select cells in Puromycin for 7 10 days very few colonies if any should be seen comparing to cloning donor vector TALE Nuclease Sample c Presence of PuroR GFP colonies indicates frequency of random integration events 2 To comfirm donor vector integration specifically at the AAVS1 target locus junction PCR can be performed using PCR primer pairs that flank the 5 AAVS1 homology arm 5 AAVS1 HA L and 3 AAVS1 homology arm 3 AAVS1 HA R 3 Protocol for Junction PCR 1 Primer sequences Primer description 5 AAVS1 Positive Control Donor Forward Primer 5 AAVS1 Positive Control Donor Reverse Primer 3 AAVS1 Positive Control Donor Forward Primer 3 AAVS1 Positive Control Donor HOPAVSHR 3R Reverse Primer The primers are provided as mixes F R primers at 10uM Vali
13. to providing our customers with high quality products If you should have any questions or concerns about any GeneCopoeia products please contact us at 301 762 0888 2013 GeneCopoeia Inc For Research Use Only 2013 GeneCopoeia Inc Trademark Genome TALER EndoFectin GeneCopoeia GeneCopoeia Inc SH 070813 10
14. ucts or deliver information obtained in service without prior written consent from GeneCopoeia This Product should be used in accordance with the NIH guidelines developed for 9 Genome TALER Human AAVSI Safe Harbor Gene Targeting Kit recombinant DNA and genetic research Use of any part of the Product constitutes acceptance of the above terms Limited Warranty GeneCopoeia warrants that the Product meets the specifications described in the accompanying Product Datasheet If it is proven to the satisfaction of GeneCopoeia that the Product fails to meet these specifications GeneCopoeia will replace the Product In the event a replacement cannot be provided GeneCopoeia will provide the purchaser with a refund This limited warranty shall not extend to anyone other than the original purchaser of the Product Notice of nonconforming products must be made to GeneCopoeia within 30 days of receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to the actual purchase price GeneCopoeia s liability does not extend to any damages arising from use or improper use of the Product or losses associated with the use of additional materials or reagents This limited warranty is the sole and exclusive warranty GeneCopoeia does not provide any other warranties of any kind expressed or implied including the merchantability or fitness of the Product for a particular purpose GeneCopoeia is committed
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