Genome-TALER™ Human AAVS1 Safe Harbor
Contents
1. Introduction to 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 provide a simple one to one code for binding to each DNA base of in the 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 Introduction to CRISPR Cas9 In the CRISPR Cas9 system the complex of a CRISPR RNA crRNA annealed to a trans activating crRNA tracrRNA is sufficient to guide the Cas9 endonuclease to a specific genomic sequence to generate a double strand break DSB in the target DNA This system can be simplified by fusing crRNA and tracrRNA sequences to produce a synthetic chimeric single guided RNA sgRNA The selected target sequence consists of a 20 bp DNA sequence complementary to the crRNA or the chimeric sgRNA followed by the trinucleotide 5 NGG 3 3 Mouse ROSA26 Safe Harbor Gene knock in Kit protospacer2. Assay for genome editing tools cutting and HR of donor vectors on samples as follows 1 ROSA26 TALENs or MCP ROSA26 CG01 positive control DC RFP SH02 Select cells in Puromycin for 7 10 days The resulting colonies should be RFP amp GFP 14 Mouse ROSA26 Safe Harbor Gene knock in Kit positive 2 Positive control DC RFP SHO02 only Select cells in Puromycin for 7 10 days after which very few colonies if any should be seen compared with Sample a The presence of PuroR RFP GFP colonies indicates random integration events 3 ROSA26 TALENs or MCP ROSA26 CG01 donor in vector DC DON SH02 Select cells in Puromycin for 7 10 days after which colonies should be GFP positive Expression of the insert may be detected by qPCR or Western blot 4 Donor in vector DC DON SH02 only Select cells in Puromycin for 7 10 days after which very few colonies if any should be seen compared with Sample c The presence of PuroR GFP colonies indicates random integration events To confirm donor vector integration specifically at the ROSA26 target locus junction PCR can be performed using PCR primer pairs that flank the 5 ROSA26 homology arm 5 ROSA26 HA L and 3 ROSA26 homology arm 3 ROSA26 HA R Protocol for Junction PCR 1 Primer sequences Primer description Primer name Primer sequence 5 ROSAS Posiive Control Donor MQPROSHR 5F EE Forward Primer 5 ROSA26 Positive Control Donor e MQPROSHR 5R See datasheet R
3. PCR primer 10M 0 2ul Zu 50X dNTP mix 10 mM of each 2 5ul 25ul 10X PCR Reaction Buffer 21 9ul 219 Nuclease free water 0 2ul Zu Taq DNA polymerase approx 5 U ul 25ul 250ul 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 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 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 ROSA26 genome editing tools 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 ROSA26
4. bGHpA EFla G 2A Puro SV40pA HAR lt _ lt _ lt 5 R primer 3 R primer 1 2kb D Figure 3 Genome CRISP mouse ROSA26 safe harbor gene knock in kit components A ROSA26 CRISPR Cas9 and donor plasmids B Knock in verification primer pairs 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 EndoFectin Plus Transfection Reagent Genecopoeia Cat EFP1003 01 02 Qiagen EndoFree Plasmid Maxi Kit Qiagen Cat 12362 Qiagen DNeasy Blood and Tissue Kit Qiagen Cat 69504 9 Proof 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 aRWN gt D Jo Mouse ROSA26 Safe Harbor Gene knock in Kit lll Example A bGH poly A mROSA26 donor control mROSA26 HA Left Flag cH pot mROSAZ6 left TALEN H ee a C m
5. gt ee bGHpA EFta T2A Puro SV40pA isang lt lt lt 5 R primer 3 R primer ad cased Figure 2 Genome TALER mouse ROSA26 safe harbor gene knock in kit components A ROSA26 TALEN and donor plasmids B Knock in verification primer pairs Genome CRISP mouse ROSA26 safe harbor gene knock in kit Cat SH ROS K200 Genome CRISP mouse ROSA26 safe harbor gene knock in kit without donor Cat SH ROS K002 Product name Qty Concentration Shipping and Storage All in one ROSA26 Shipped at room MCP ROSA26 CG01 sgRNA Cas9 Dns expression clone temperature Stored at 20 C Shipped at room DC DON SH02 ROSAZ26 donor vector 10 ug 500 ng ul temperature Stored at 20 C DC RFP SH02 ROSA26 RFP control 10 500 ng ul Sinopec ek roem Hg temperature Stored at 20 C e 200 Shipped at room MQPROSHR 5 5 HR primer pair 10 uM temperature Stored at 20 C S 200 Shipped at room MQPROSHR 3 3 HR primer pair 10 uM temperature Stored at 20 C DC DON SH02 only comes with SH ROS K200 kit ROSA26 knock in ORF donor clones can be customized and purchased separately A CRISPR Cas9 and donor plasmids mROSAZ6 sgRNA T7 10 0W01d OPAS mROSA26 sgRNA Cas9 clone Sv40 PolyA Mouse ROSA26 Safe Harbor Gene knock in Kit mROSA26 donor control mROSA26 HA Left mROSA26 HA Right mROSA26 HA Left mROSA26 HA Right B Knock in verification PCR primers 5 F primer 3 F primer e Chr 19
6. _ bGHpA TA Puro Map MRR lt lt 5 R primer ame lt lt 3 R primer pop 16 Mouse ROSA26 Safe Harbor Gene knock in Kit Tech Note 1 If the junction PCR band is weaker than 5 junction PCR band it is likely that the amplification efficiency for the 3 junction region is lower due to the nature of the chromosomal structure modification and sequence around that region 2 One positive in junction PCR is sufficient to confirm the integration 3 Though rare it is possible that random integration can coexist with ROSA26 specific integration Southern blotting can be used to detect coexisting random integration The method is described in http bloodjournal hematologylibrary org content 11 7 21 5561 fu bim VI References 1 Zou J et al 2009 Gene targeting of a disease 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 R 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 R globin transgene expression in thalassemia induced pluripotent stem cells Nat Biotechnol 2011 29 1 73 8 5 Lombardo A
7. adjacent motif PAM which is recognized by the Cas9 and essential for cleavage This RNA guided DNA recognition mechanism of CRISPR Cas9 provides a simple but powerful tool for precision genome engineering The GeneCopoeia Genome TALER mouse ROSAZ26 safe harbor gene knock in kit is designed to specifically transfer your gene of interest selection marker or other genetic element from a donor plasmid into the ROSA26 safe harbor site on mouse chromosome 6 via TALEN mediated homologous recombination HR HR is a natural DNA repair mechanism that occurs in response to DNA double strand break DSB This DSB is created by a ROSA26 specific TALEN The GeneCopoeia Genome CRISP mouse ROSAZ26 safe harbor gene knock in kit is designed to specifically transfer your gene of interest selection marker or other genetic element from a donor plasmid into the ROSA26 safe harbor site on mouse chromosome 6 via CRISPR Cas9 mediated homologous recombination HR HR is a natural DNA repair mechanism that occurs in response to DNA double strand breaks DSB This DSB is created by an ROSA26 specific CRISPR Cas9 system Advantages Safe integration Designated ROSA26 mouse genome safe harbor integration site ensures transcription competency of the transgenes and presents no known adverse effect on cells Specific targeting TALEN mediated DNA DSBs at the ROSA26 site stimulate homologous recombination dramatically for transgene integration Single copy numbe
8. keen ES Fees ii i BS ee SSES ES ES SS EES SES eme Cen Lem emeng en emer rom EE Figure 6 Illustration of serial dilution 2 Add 200ul cell suspension to well A1 Mix 100uI from AT with the medium in well B1 Avoid bubbles Continue this 1 2 dilution through column 1 Add 100u of medium back to column 1 so that wells A1 through H1 contain 200uIl 3 Mix cells and transfer 100uI of cells from column 1 into column 2 Mix by gently pipetting Avoid bubbles Repeat these 1 2 dilutions through the entire plate Bring the final volume to 200ul by adding 100ul of medium to all but the last column of wells 4 Incubate plates undisturbed at 37 C 5 Cells will be observable via microscopy over 3 days and be ready to score in 5 8 days depending on the growth rate of cells Mark each well on the cover of the plate indicating which well contains a single colony These colonies can later be subcultured from the well into larger vessels Tech Note 1 Adding 4000 cells in well A1 2x104 cells ml is a good starting concentration Increase the concentration for more difficult to grow cell lines 2 If the reporter gene is fluorescent determine which of these colonies express it If the reporter gene is not observable you will have to wait until later in the culture process 3 Label each well with a single colony using a unique identification number and record this number on the plate and in your notebook Validation of HR recombinant cells 1
9. Gen eCop e ajg Expressway to Discovery Genome TALER amp Genome CRISPR Mouse ROSA26 Safe Harbor Gene Knock in Kit Catalog SH ROS K100 Catalog SH ROS K000 Catalog SH ROS K200 Catalog SH ROS K002 User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2014 GeneCopoeia Inc Mouse ROSA26 Safe Harbor Gene knock in Kit USER MANUAL Genome TALER Mouse ROSAZ26 Safe Harbor Gene Knock in Kit Genome CRISP Mouse ROSA26 Safe Harbor Gene Knock in Kit I Introduction Il Content and Storage lll Example IV Overview of Safe Harbor Integration V Critical Steps VI References VII Related Services VIII Licensing and Warranty Statement I Introduction Safe gene targeting Genome modification 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 predete
10. ROSA26 HA Left mROSA26 HA Right mROSA26 sgRNA T7 Control z Red flourescene Green flourescene Phase contrast exposure time 1s exposure time 1s exposure time 1s d S S mROSA26 sgRNA Cas9 clone Sv40 PolyA put of Jame C 5 F primer Mouse genome 3 F primer _ bGHpA EFta P o Puro o O MRE lt lt lt 5 R primer 3 R primer 1 1 pc aed D Figure 5 Mouse genome safe harbor ROSA26 gene targeting A ROSA26 RFP control plasmid DC RFP SH02 800 ng was ROSA26 Negative CRISPR Cas9 Control co transfected with ROSA26 all in one bp sgRNA Cas9 expression clone 600 ng or control of only control plasmid 3000 2500 DC RFP SH02 800 ng transfected into 2000 mouse Neuro2a cells in a 6 well pate eo B 48 hr post transfection the cells were 1000 split 1 10 into a new 6 well pate and 750 selected against 1 0 ug ml of puromycin 500 The images were taken after two weeks of selection Few colonies left in the wells transfected with only ROSA26 RFP Primer Set GCI control C D PCR primers designed to amplify the HR junction were used to verify the specific and successful integration Mouse ROSA26 Safe Harbor Gene knock in Kit IV Overview of Safe Harbor Integration Plasmid propagation in E coli highly recommended Cloning into empty DC DON SH02 vector TALEN mediated ROSA26 safe harbor knockin Co transfection of ROSA26 TALEN and knockin clone control highly reco
11. ROSA26 right TALEN i co mia lt TD B Sample Phase contrast exposure time 1s Green flourescene exposure time 1s Red flourescene exposure time 1s Control bh Phase contrast exposure time 1s Green flourescene exposure time 1s Red flourescene exposure time 1s 5 F primer Mouse genome 3 F primer CMV 4 GOI bGHpA EFia E lt 5 R primer 3 R primer ame D ROSA26 Negative TALEN Control 5 3 5 3 M Bp 3000 2500 2000 1500 1250 1000 750 500 Primer Set GCI 1 Bip Figure 4 Mouse genome safe harbor ROSA26 gene targeting A ROSA26 RFP control plasmid DC RFP SH02 800 ng was co transfected with ROSA26 TALEN pair 600 ng for each or control only DC RFP SH02 800 ng transfected into mouse Neuro2a cells in a 6 well pate B 48 hr post transfection the cells were split 1 10 into a new 6 well pate and selected against 1 0 ug ml of puromycin The images were taken after two weeks of selection Few colonies left in the wells transfected with only ROSA26 RFP control C D PCR primers designed to amplify the HR junction were used to verify the specific and successful integration Mouse ROSA26 Safe Harbor Gene knock in Kit A B ER poly A T2A Soch Sample Red flourescene Green flourescene Phase contrast exposure time 1s exposure time 1s exposure time 1s 2 mROSA26 donor control JE D m
12. TALENs or MCP ROSA26 CG01 positive control DC RFP SH02 12 Mouse ROSA26 Safe Harbor Gene knock in Kit b Positive control DC RFP SH02 only c ROSA26 TALENs or MCP ROSA26 CG01 donor in vector DC DON SH02 d Donor in vector DC DON SH02 only 2 The next day prepare transfection complexes of genome editing tool plasmids and donor plasmids using suitable transfection reagents according to the manufacturer s instructions Leave the transfection complexes on the cells to react for gt 6 hours Example For Neuro2A cells using EndoFectin Plus Transfection Reagent transfect 0 5yg of each TN ROSA26 L and TN ROSA26 R vectors ug total and 1pg of donor vector Tech Notes 1 Since transfection efficiencies vary across different cell lines we recommend optimizing the input of genome editing tool plasmids to donor vectors for best results We recommend starting with a 1 1 ratio e g 1g of donor HR plasmid 0 5ug of each TALE Nuclease plasmid or 1ug of MCP ROSA26 CG01 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 A 24 hours post transfec
13. ceipt 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 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 2014 GeneCopoeia Inc For Research Use Only 2014 GeneCopoeia Inc Trademark Genome TALER Genome CRISP SH 051214 EndoFectin GeneCopoeia GeneCopoeia Inc 20
14. et al 2011 Site specific integration and tailoring of cassette design for sustainable gene transfer Nat Methods 2011 8 10 861 9 17 Mouse ROSA26 Safe Harbor Gene knock in Kit VII Related Services Stable cell line services GeneCopoeia offers monoclonal stable cell line service with customized TALEN or CRISPR Cas9 mediated genome modifications Cell banking service is also available TALEN CRISPR Stable Cell Line Development Services ee e Standard transfection 4 4 Safe Harbor clone E E eel optional H EE 4 to rule out RI e Select de with single or double allele modifications to make cell bank 4 e Select best clone with single or double modifications to make master cell bank e PCR verification for one vial of MCB 18 Mouse ROSA26 Safe Harbor Gene knock in Kit Transgenic mouse services GeneCopoeia offers transgenic mice with customized TALEN or CRISPR Cas9 mediated genome modifications TALEN and CRISPR Cas9 Mediated Genome Modifications Overview of genome editing by TALEN and CRISPR Cas9 TALEN CRISPR Cas9 mami o ent NHE HR ke D zg SSE One step generation of mice with genome modifications TALEN o amp K sgRNA donor DNA eg e NHEJ or HR fg j Mi y injection d amr Gr Blastocyst Mutant 19 Mouse ROSA26 Safe Harbor Gene knock in Kit Vill Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of the Geno
15. everse Primer 3 ROSA26 Positive Control Donor MQPROSHR 3F See datasheet Forward Primer 3 ROSA26 Positive Control Donor Reverse Primer MQPROSHR 3R See datasheet The primers are provided as mixes F R primers at 10uM Validation of either the 5 or 3 homology arms for donor integration is usually sufficient however both arms can be done for additional confirmation 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 the protocol recommended by the manufacturer b Perform junction PCR PCR reaction below 15 Mouse ROSA26 Safe Harbor Gene knock in Kit Reagent Genomic DNA 60 100ng ul 10uM 5 or 3 ROSA26 PCR Primer Mix 5X UltraPF Buffer Mg free 10 mM dNTPs 20mM MgSO 4 UltraPF 5U ul PCR grade distilled water Total 98 C 5min 98 C 20sec 55 C 30sec 35 cycles 72 C 1min 72 C 7min Hold at 4 16 C TALEN cutt positive Positive control control donor donor only Tul Jul Jul 5ul 5ul Ubu 0 5ul 2 5ul 2 5ul 0 25ul 0 25ul 14 75ul 14 75ul 25ul 25ul ROSA26 Negative CRISPR Cas9 Control 5 3 5 3 M bp 1250 Primer Set GCI Run the PCR reaction on a 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 Mouse genome 3 F primer
16. me TALER mouse ROSA26 Safe Harbor Gene Knock in Kit amp Genome CRISP mouse ROSA26 Safe Harbor Gene Knock in 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 products 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 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 re
17. mmended Antibiotic selection or cell sorting to enrich for clones with donor integration highly recommended Isolation of single colonies Validation of HR recombinant cells Screen positive clones by junction PCR Southern blotting to eliminate clones with random donor integration highly recommended Monoclonal master cell bank preparation and storage Optional CRISPR Cas9 mediated ROSA26 safe harbor knockin Co transfection of ROSA26 CRISPR Cas9 and knockin clone control highly recommended Antibiotic selection or cell sorting to enrich for clones with donor integration highly recommended Isolation of single colonies Validation of HR recombinant cells Screen positive clones by junction PCR Southern blotting to eliminate clones with random donor integration highly recommended Monoclonal master cell bank preparation and storage Mouse ROSA26 Safe Harbor Gene knock in Kit V Critical Steps 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 ROSA26 TALENs ROSA26 CRISPR Cas9 and plasmids in the DC DON SH02 vector we suggest plating 50 200ul of transformed cells on fresh LB Ampicillin plates 50ug ml Incubate the plates at 37 C overnight Inoculate colonies from the transfo
18. r Known copy number of the transgene ensures predictable expression levels simplifies phenotype interpretation and prevents transgene silencing Compatible knock in ORFs Over 20 000 sequence verified mouse ORFs are compatible for transgene donor DNA design Mouse ROSA26 Safe Harbor Gene knock in Kit ll Content and storage Genome TALER mouse ROSA26 safe harbor gene knock in kit Cap SH ROS K100 Genome TALER mouse ROSAZ26 safe harbor gene knock in kit without donor Cat SH ROS K000 ROSA26 left TALEN 10 ug 500 ng l ROSA26 right TALEN 10 ug 500 ng l ROSAZ26 donor vector 10 ug 500 ng ul DC RFP SH02 ROSA26RFP control 10 ug 500 ng l 5 HR primer pair 200 reactions 10 uM 3 HR primer pair 200 reactions 10 uM 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 DC DON SHO02 only comes with SH ROS K100 kit ROSA26 knock in ORF donor clones can be customized and purchased separately A TALEN and donor plasmids bGH poly A T2A PuroR mROSA26 donor control v Alod mg mROSA26 HA Left mROSA26 HA Right mROSA26 HA Left B Knock in verification PCR primers mROSA26 HA Right Mouse ROSA26 Safe Harbor Gene knock in Kit 5 F primer Mouse genome 3 F primer
19. rmation and grow them at 37 C overnight in 200mlI of LB media containing 50ug ml of Ampicillin 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 DC DON SH02 vector 1 Ligation 1 Digest and gel purify the vector plasmid Dilute it to 10ng ul 2 Get up 10ul ligation reaction for each control and test sample Volume Item 1 0 ul Digested DC DON SH02 empty vector 7 0 ul DNA insert 30 50 ng or water control 1 0 ul 10X T4 DNA ligase buffer 1 0 ul T4 DNA Ligase 40 U l 10 0 pl Total Reaction Volume 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 11 Mouse ROSA26 Safe Harbor Gene knock in Kit 2 Prepare a PCR Master Mix with PCR primers flanking the insert 1rxn 10 rxn Composition O 1p Ju 5 PCR primer 10M O 1pl Ju 3
20. rmined and safe site in a genome ROSA26 also known as ROSA D geo26 locus in mice genome is first found in chromosome 6 in one particular strain of mice named ROSA D geo26 expressed H galactosidase from a randomly inserted transgene at high levels uniformly in nearly all tissues examined This locus expresses one coding transcript and two noncoding transcripts and only the non coding transcripts are disrupted by the insertion While pups homozygous for the insertion are born at slightly lower frequency than heterozygous pups homozygotes appeared to develop normally and were fertile So the Rosa26 locus has since been used as a transgene insertion site that causes no apparent adverse effects on fitness and permits stable gene expression The GeneCopoeia ROSA26 specific TALEN or CRISPR Cas9 systems can generate a DNA double strand break DSB in ROSA26 on mouse chromosome 6 stimulating natural DNA repair mechanisms In the presence of ROSA26 ORF knockin clones homologous 2 Mouse ROSA26 Safe Harbor Gene knock in Kit recombination HR occurs resulting in integration of the DNA fragment from the ORF knockin clone into the safe harbor locus gt ROSA26 locus Site specific genome editing tools DSB Chr ORF Donor Clone So os EEN ORF knock in ROSA26 locus J HR Chr DEE EECH R Analysis of GENE knocked in Figure 1 Illustration of genome editing tool mediated transgene integration at the mouse safe harbor ROSAZ26 site
21. tion 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 Neuro2A 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 5yg ml that kills gt 90 of cells after 48hours of selection is the correct dose for the cells being selected Clonal isolation of cell lines Serial dilution is widely used to isolate single clones with desired modifications followed by an expansion period to establish a new clonal cell line Like most clonal isolation methods there is no guarantee that the colonies arose from single cells A second round is advised to increase the likelihood of clonal isolation Also it is worth noting that cell types can vary substantially in their responses to single cell isolation therefore literature specific to the cell type of interest should be consulted 13 Mouse ROSA26 Safe Harbor Gene knock in Kit 1 Fill each well of a sterile 96 well plate with 100uI of medium except for well A1 which should remain empty pe ee RE PS een e ee ES Fe E
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