Genome-TALER™ TALEN and TALE-TF Products
Contents
1. arm L Recombination arm L Recombination arm L Genome TALER TALEN and TALE TF Products and Services Sv40 PA pDonor D0O2 y we UO eUIqUIODey Recombination arm L y we uojeuqwoovay Recombination arm L Sv40 PA y we uoneulqwoIasy Recombination arm L Sv40 PA y we UO EUIqUIO ay Recombination arm L y wue UONeUIquiosey Figure 6 Maps of donor vectors 14 y we UO eUIqUIO ey y we uoneuiqwovsy y we uopeulquwooay y wue uonpeujqwov y Genome TALER TALEN and TALE TF Products and Services Stable cell line services GeneCopoeia offers monoclonal stable cell line service with customized TALEN mediated genome modifications Cell banking service is also available TALEN CRISPR Stable Cell Line Development Services e Clone Standard cell line or Custom cell line e Standard transfection e Selection antibiotics or eGFP e PCR screening of stable clones e Pick single colonies Safe Harbor clone only optional e Southern blot to rule out RI e Select clones 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 Genome TALER TALEN and TALE TF Products and Services VIII Limited Use License and Warranty Limited Use License Following terms and conditions apply to use of the Genome TALER TALEN and TALE TF products and servic2. specific genome editing For gene knockout knockin mutagenesis activation repression and more Flexible TAL effector design of binding and functional domains such as TALEN and TALE TF Genome TALER TALEN and TALE TF Products and Services ll Products and Services Services Validation Donor clone services see appendix Stable cell line services see appendix Transgenic mouse services Surrogate reporter assay T7 endonuclease assay Donor clone design and construction Monoclonal colony Cell bank Transgenic mouse Description Plasmid level functional validation Detects activities of genome editing tools by observing the expression level of a surrogate reporter gene Chromosomal level functional validation Detects the presence of indels created by TALEN mediated NHEJ repair at the specific target site of the chromosome Customized plasmids designed to specifically transfer your gene of interest selection marker or other genetic elements into targeted sites through homologous recombination HR induced by our TALEN We offer various donor vector choices with different selection markers and genetic elements built in for your experiment purpose Monoclonal stable cell line with TALEN mediated genome modifications Creation of a cell bank of monoclonal stable cell line with TALEN mediated genome modifications Transgenic mice with TALEN mediated genome modifications Genome T
3. AACt method Option B Measuring TALEN cutting efficiency using mismatch cleavage assay TALEN modified DNA will have a few bases of sequence deletion near the TALEN cut site because of NHEJ exonuclease activity We recommend using the Surveyor mutation detection kit for standard gel electrophoresis Transgenomic cat no 706025 for this assay Alternatives include the Cel1 T7 mung bean and S1 nucleases The Surveyor procedure is carried out according to the manufacturer s instructions and is described in greater detail in the Surveyor manual We provide brief details here Genome TALER TALEN and TALE TF Products and Services 24 or 48 hr post transfection collect cells to extract genomic DNA PCR amplify the region surrounding the sgRNA target site Check the PCR result by running 5 ul of PCR product on a 2 agarose gel For all templates it is important to make sure that there is only a single band corresponding to the intended product for the primer pair The size of this band should be the same as calculated from the distance between the two primer annealing sites in the genome CRITICAL STEP If multiple amplicons are generated from the PCR redesign the primers and reoptimize the PCR conditions to avoid off target amplification In difficult cases in which a single band product cannot be achieved it is acceptable to gel extract the correct length band before proceeding with heteroduplex reannealing and Surveyor nuclease diges
4. ALER TALEN and TALE TF Products and Services lil Examples A B kDa 3 b 250 150 i 1 Left TALEN Niis 2 Right TALEN 100 i 3 Cells alone 70 E eo 55 a EX EGFP Lv105 200 ng Control Plasmid 800 ng b EX EGFP Lv105 200 ng EGFP TALENs 800 ng Figure 3 TALENs knockdown eGFP expression A eGFP TALENs expression validation 80 confluence HEK293T cells were transfected with 0 8 ug plasmid per well in a 6 well plate The cells were harvested 48 hrs post transfection 1 20th of the cell lysate per well was analyzed for western blot using anti Flag antibody in a SDS PAGE 8 resolve gel with the untransfected cell lysate as the negative control B TALENs knockdown eGFP expression HEK293T cells in a 6 well plate were co transfected with EX EGFP Lv105 and TALEN plasmids or control plasmid EGFP expression was visualized under a microscope Nikon Eclipse Ti exposure time 600ms 48hrs post transfection 25 Figure 4 NTF3 TALE TF regulates 20 endogenous NTF3 transcription Endogenous NTF3 transcription activation 15 by TALE TF HEK 293T cells transfected with the NTF3 TALE TF 6 well plate 1 ug m plasmid per well exhibited a 17 fold e 10 increase in the amount of NTF3 mRNA compared to cells transfected with an 5 empty vector Measurements were performed in triplicate 0 Empty Vector NTF3 TALETF Genome TALER TALEN and TALE TF Products and Services IV Overview of Genome Editing U
5. Genome TALER TALEN and TALE TF Products and Services Genome TALER TALEN and TALE TF Products and Services User Manual GeneCopoeia Inc 9620 Medical Center Drive 101 Rockville MD 20850 USA 301 762 0888 866 360 9531 inquiry genecopoeia com www genecopoeia com 2014 GeneCopoeia Inc Genome TALER TALEN and TALE TF Products and Services USER MANUAL Genome TALER TALEN and TALE TF Products and Services Introduction to TAL effectors ll Related Services lll Examples IV Overview of Genome Editing Using TALEN and TALE TF V Critical Steps VI References VII Appendix VIII Licensing and Warranty Statement l Introduction Transcription activator like TAL effectors are proteins secreted by Xanthomonas bacteria when they infect plants These proteins can activate the expression of plant genes by recognizing and binding 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 are hyper variable There appears to be a simple one to one code between these two critical amino acids in each repeat and each DNA base in the target sequence e g NI A HD C NG T and NN G or A Recent work has demonstrated that the NH RVD has greater specificity and comparable affinity for G compared with NN Therefore the NN RVD has been replaced for G recognition by NH GeneCopoeia also
6. Vol 39 No 12 e82 doi 10 1093 nar gkr218 Li T et al Modularly assembled designer TAL effector nucleases for targeted gene knockout and gene replacement in eukaryotes Nucleic Acids Research 2011 Vol 39 No 14 6315 6325 doi 10 1093 nar gkr188 Zhang F et al Programmable Sequence Specific Transcriptional Regulation of Mammalian Genome Using Designer TAL Effectors Nat Biotechnol 2011 February 29 2 149 153 doi 10 1038 nbt 1775 12 Genome TALER TALEN and TALE TF Products and Services Vil Appendix Donor services GeneCopoeia offers customized donor clone design and construction services Donor clones are customized plasmids designed to specifically transfer your gene of interest selection marker or other genetic elements into a target site via HR mediated repair of DSBs induced by site specific TALENs or CRISPR Cas9 Donor vectors are available with several options for selection markers and genetic elements to meet your experimental needs Donor vector types Reporter Selection Vector Promoter LoxP Site Gene Marker pDonor D01 EFa copGFP Puromycin N A pDonor D02 CMV copGFP Neomycin N A pDonor D03 CMV N A Neomycin N A pDonor D04 CMV N A Puromycin N A pDonor D05 EFa N A Neomycin N A pDonor D07 EFa1 copGFP Puromycin TK Loxp pDonor D08 CMV copGFP Neomycin TK Loxp pDonor D09 EFa1 N A Puromycin TK Loxp pDonor D10 CMV N A Neomycin TK Loxp 13 Recombination arm L Revoind ination arn Recombination
7. asmid to transfection reagent for best results For optimal results we recommend complexing of DNA with transfection reagent in serum and antibiotic free media and cells growing in complete media For hard to transfect cells e g primary stem hematopoietic it may be advisable to utilize a nonpassive transfection method such as NucleoFection Lonza or Neon system Life Technologies Please follow recommended transfection guidelines provided by the manufacturer for specific cell type s being transfected 8 Genome TALER TALEN and TALE TF Products and Services 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 Allow cells to recover for 24 hours 4 Begin antibiotic selection 48 hours post transfection We recommend optimizing concentration of antibiotic for best results Tech Note Establishing a kill curve on untransfected cells can determine the effective working antibiotic concentration for a target cell line The concentration of antibiotic that kills gt 90 of cells after 48hours of selection is the correct dose for the cells being selected Example For HEK293T cells using EndoFectin reagent transfect TALEN or TALE TF 1 2 5 Plate cells Plate HEK293T cells onto 6 well plates 24 h before transfection The number of cells plated in each well should be d
8. ded before your gene targeting experiment Plasmids can be transformed using standard conditions suitable in any RecA and EndA E coli competent cells For transformation of TALE product plasmids 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 transformation and grow them at 37 C overnight in 200ml of LB media containing SOyug ml Ampicillin Use an endotoxin free plasmid DNA maxiprep kit to extract plasmid DNA after overnight growth To confirm the integrity of the amplified plasmids we recommend restriction digestion analysis or direct sequencing B Characterization of TALE TF or TALEN modified cells Option A Measuring TALE TF transcriptional activation using qRT PCR For TALE TFs qRT PCR quantitatively measures the increase in transcription driven by the TALE TF GeneCopoeia provides validated qPCR primers for most genes in the human genome Full services covering RNA extraction reverse transcription quantitative PCR and data analysis are also available from GeneCopoeia There are a wide variety of qRT PCR protocols We provide brief details here 1 24 or 48hr post transfection collect cells to extract total RNA 2 Measure the RNA concentration using a UV spectrophotometer 3 Perform reverse transcription to get CDNA 4 Perform quantitative PCR 5 Analyze data and calculate the level of gene activation using the
9. es 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 receipt of the Product GeneCopoeia s liability is expressly limited to replacement of Product or a refund limited to
10. etermined so that they are 70 80 confluent at the time of transfection Prepare the DNA Opti MEM mix Option A mix 2 0ug of TALE TF plasmid DNA with 50ul of Opti MEM Notes include controls e g a reporter plasmid or mock transfection to monitor transfection efficiency and cell health respectively Option B mix 1 0ug of each Left TALEN and Right TALEN 2pg total with 50ul of Opti MEM Notes control transfections can be done by omitting one or both of the TALENS Include controls e g a reporter plasmid or mock transfection to monitor transfection efficiency and cell health respectively Prepare the EndoFectin Opti MEM solution Dilute 6ul of EndoFectin Plus with 50ul of Opti MEM Mix the solution thoroughly at room temperature Prepare DNA EndoFectin complex Add the diluted EndoFectin reagent drop wise to the DNA solution while gently vortexing the DNA containing tubes Note Do not reverse the addition sequence Incubate the mixture for 10 25 minutes at room temperature to allow the DNA EndoFectin complex to form Transfect cells Add the DNA EndoFectin complex directly to each well and gently swirl the plates dishes Genome TALER TALEN and TALE TF Products and Services D Validation of TALEN modified and HR recombinant cells 1 To confirm donor vector integration specifically at a target site junction PCR can be performed using PCR primer pairs that flank the 5 homology arm and 3 h
11. gration can coexist with site specific integration Negative selection can be used to detect coexisting random integration E 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 1 Fill each well of a sterile 96 well plate with 100ul of medium except for well A1 which should remain empty at E Figure 5 Illustration of serial dilution 2 Add 200ul cell suspension to well A1 Mix 100ul from A1 with the medium in well B1 Avoid bubbles Continue this 1 2 dilution through column 1 Add 100ul of medium back to column 1 so that wells A1 through H1 contain 200ul 3 Mix cells and transfer 100ul 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 11 Genome TALER TALEN and TALE TF Products and Services Cells will be observable via m
12. icroscopy 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 2 3 VI Adding 4000 cells in well A1 2x104 cells ml is a good starting concentration Increase the concentration for more difficult to grow cell lines 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 Label each well with a single colony using a unique identification number and record this number on the plate and in your notebook References Boch J et al Breaking the code of DNA binding specificity of TAL type Ill effectors Science 2009 326 5959 1509 12 Moscou M et al A simple cipher governs DNA recognition by TAL effectors Science 2009 326 5959 1501 Christian M et al Targeting DNA Double Strand Breaks with TAL Effector Nucleases DOI 10 1534 genetics 110 120717 Morbitzera R et al Regulation of selected genome loci using de novo engineered transcription activator like effector TALE type transcription factors www pnas org cgi doi 10 1073 pnas 1013133107 Cermak T et al Efficient design and assembly of custom TALEN and other TAL effector based constructs for DNA targeting Nucleic Acids Research 2011
13. omology arm 2 Protocol for junction PCR 1 Primers should be diluted to 10uM before use 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 Genomic DNA 60 100ng ul 1ul 1 ul 10uM 5 or 3 PCR Primer Mix 5 X UltraPF Buffer Mg free 10 mM dNTPs 0 5ul 0 5ul 20mM MgSO 2 5ul 2 5ul UltraPF 5U ul 0 25ul 0 25ul PCR grade distilled water 14 75ul 14 75ul Total 25ul 25ul 98 C Smin 98 C 20sec 95 C 30sec 35 cycles 72 C 1min 72 C 7min Hold at 4 16 C 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 depend on design 10 Genome TALER TALEN and TALE TF Products and Services Tech Note 1 The 3 junction PCR band and 5 junction PCR band may differ in brightness because the amplification efficiency may be different 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 inte
14. sing TALEN and TALE TF Plasmid propagation in E coli highly recommended TALEN or TALE TF chromosomal validation optional HR based knockin Genome regulation with a donor through transactivation For mutagenesis tagging Transfection of TALE TF plasmids knockin selection marker etc Antibiotic selection to Antibiotic selection to Antibiotic selection or enrich for transfected enrich for transfected cell sorting to enrich for cells cells transfected cells highly recommended highly recommended highly recommended NHEJ based knockout without a donor Co transfection of Co transfection of TALEN and donor TALEN plasmids plasmids 2 round of antibiotic selection or cell sorting to enrich for clones with donor integration highly recommended TK selection to eliminate clones with random donor integration optional Isolation of single colonies Isolation of single colonies Validation of TALEN Screen positive clones modified and HR Validation of TALE TF by PCR and sequencing recombinant cells Screen transactivation by qPCR or other methods e g positive clones by junction T7 Endonuclease PCR or other methods Monoclonal master cell bank preparation and storage Monoclonal master cell bank preparation and storage Genome TALER TALEN and TALE TF Products and Services V Critical Steps A Plasmid propagation We recommend propagating the plasmids provi
15. 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 EndoFectin GeneCopoeia GeneCopoeia Inc TN 130813 16
16. tion DNA heteroduplex formation At this point the amplified PCR product includes a mixture of both TALEN modified and unmodified genomic DNA Place 300 ng of the PCR product in a thermocycler tube and perform the cross hybridization Surveyor Nuclease S digestion To treat the cross hybridized homo and heteroduplexes with Surveyor Nuclease S to determine TALEN cleavage efficiency C Transfection of TALE TF or TALEN into target cells Option A if you are using TALE TF for transcriptional modulation or option B if you are using TALEN for testing nuclease activity 1 Plate 100 000 to 300 000 cells well in a 6 well plate according to established recommended conditions for the cell type s being transfected Scale up and down the culture if needed 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 The next day prepare transfection complexes of TALEN and TALE TF using a suitable transfection reagent according to the manufacturer s recommended instructions Leave the transfection complexes on the cells to react for gt 6 hours Option A 2 0ug of TALE TF Option B 1 0ug of LEFT TALEN 1 0ug of RIGHT TALEN Notes including appropriate controls according to your experiment Tech Notes 1 Since transfection efficiencies vary across different cell lines we recommend optimizing the input of pl
17. uses the N RVD for recognition of 5 methyl cytosine Figure 1 34aa repeat modules Effector domain TTTATTCCCTGACC ot Wa eos gee wa a ea s DS PE aat iai o esas a e a LTPEQVVAIASNGGGKQALETVQRLLPVLCQAHG Variable Diresidues m o o N O o pT c A GA j c se Figure 1 Top Schematic of a TAL effector Bottom Typically used RVD recognition code Genome TALER TALEN and TALE TF Products and Services TAL effectors have been utilized to create site specific gene editing tools by fusing target sequence specific TAL effectors to nucleases TALENSs transcription factors TALE TFs and other functional domains Figure 2 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 Unlike zinc fingers nucleotide triplet recognition TAL effector domains recognize single nucleotides which allow researchers to be able to specifically target whatever sequence they want o Functional Domain DNA Binding Domain Left TALEN Functional Domain Transcription Complex DNA Binding Domain Activate Transcription rd TALE TF Gene of Interest 3 5 Figure 2 Top Typical TALEN design strategy Bottom Typical TALE TF design strategy Advantages Target any gene in any cell Highly sequence
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