GenTarget`s EcoTMPlasmid DNA Miniprep Kit User Manual
Contents
1. 7930 Arjons Drive Suite B bpn 77 21 ine Fax San Diego CA 92126 Phone 858 6788683 800 3804198 Email orders gentarget com Humanized Cas9 endonuclease expression lentivirus for CRISPR lentiviral particles in vivo ready Cat Product Name Amounts LVP681 CMV h Cas9 Puro lentiviral 200ul 5 x 105 IFU mL particles Lvpesi pps CMV h Cas9 Puro lentiviral 200ul 5 x 107 IFU mL particles in vivo ready LVP682 CMV h Cas9 Bsd lentiviral 200ul 5 x 108 IFU mL M particles Lypes2 pgs CMV h Cas9 Bsd lentiviral 200ul 5 x 107 IFU mL T particles in vivo ready LVP683 CMV h Cas9 Neo lentiviral 200ul 5 x 108 IFU mL EE particles Lvpess pgs CMV h Cas9 Neo lentiviral 200ul 5 x 107 IFU mL particles in vivo ready LVP678 CMV h Cas9 GFP Puro 200ul 5 x 108 IFU mL lentiviral particles LvPeze pgs CMV h Cas9 GFP Puro 200ul 5 x 107 IFU mL CMV h Cas9 RFP Puro particles in vivo ready LVP679 e ndi paddo 200ul 5 x 10 IFU mL j CMV h Cas9 RFP Puro 7 LVP679 PBS lentiviral particles in vivo ready 2000 ete AAL paan CMV h Cas9 GFP Bsd lentiviral joo 5 x 105 IFU mL particles Lvpeso pas CMV h Cas9 GFP Bsd lentiviral 5594 5 x 107 IFU mL particles in vivo ready ezn CMV h Cas9 RFP Bsd lentiviral 200ul 5 x 105 IFU mL S particles m CMV h Cas9 RFP Bsd le2. the genomic editing via HDR mechanism The double stranded donor DNA cassette can be provided from DNA fragment synthesized or use a linearized donor vector Cas9 tracrRNA Genomic DNA crRNA NGG PAM bd LHA RHA Donor DNA GenTarget s Cas9 expression lentiviruses GenTarget is proud to offer the standalone Cas9 expression lentivirus products The ready to use Cas9 lentivirus are produced from our proprietary high titer lentivectors that express a nuclear penetrating humanized wild type Cas9 gene The Cas9 enzyme is driven by different promoters with a variety of antibiotic selection markers see the core expression vector map scheme above providing you an easy delivery for cas9 expression in almost all cell types included the hard to transfected cell types primary cells and non dividing cells which makes the gene editing possible in all cell types With using the ready to use Cas9 lentivirus you can simply synthesize the targeting expression cassette U6 H1 crRNA tracrRNA or construct the guild vector gRNA by clone the target specific crRNA tracrRNA into the a desired Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 4 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 ell arue ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com gRNA vector without cas9 cassette Note GenTarget pro
3. you first select a suitable target sequence the crRNA region see online tools for target selection below and then synthesize and anneal the crRNA oligos and clone it into the guild vector which transcripts the target specific crRNA tracrRNA sequence 2 Cas9 endonuclease The co existence of the gRNA sequence with Cas9 enzyme leads to the formation of a gRNA Cas9 complex that will bind to and cleave the corresponding genomic DNA target sequence In some cases the Cas9 and the gRNA is made in one vector So call One vector system or All in one vector However the separating Cas9 expression and guild gRNA into two vectors provides more flexibility in genomic editing because the Cas9 can be pre made like GenTargt s Cas9 expression lentivirus which makes it easier to simply construct the desired gRNA vectors Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 3 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 eli arde ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com 3 The donor DNA sequence knock In For genomic modification application a double strand repair DNA is required after the Cas9 creates the double stranded breaks at desired genomic loci The donor DNA provides the desired sequence insertion that flanked by the gene loci s homology sequences left homologous arm LHA and right homologous arm RHA for
4. Considerations for Research with Lentiviral Vectors Link Warranty This product is for research use only It is warranted to meet its quality as described when used in accordance with its instructions GenTarget disclaims any implied warranty of this product for particular application In no event shall GenTarget be liable for any incidental or consequential damages in connection with the products GenTarget s sole remedy for breach of this warranty should be at GenTarget s option to replace the products Related Products GenTarget s pre made lentivirus product catagory Product Product Description Category please click category name to see product s pages Human Premade lentivirus expressin a human mouse or rat gene with mouse or rat RFP Blastididin fusion dual markers ORFs Fluorescent Preamde lentivirus express human codon optimized fluorescent markers protein GFP RFP CFP BFP YFP Luciferase Premade lentivirus for all kinds of luciferase protein expression expression firefly and Renilla with different antibiotic selection markers CRE Premade lentivirus for expressing nuclear permeant CRE recombinase recombinase with different flurescent and antibiotic markers LoxP Premade lentivirus expressing LoxP GFP Stop LoxP RFP ColorSwitch cassette used to monitor the CRE recombination event in vivo TetR Premade lentivirus expressin TetR tetracycline regulator protein inducible t
5. the negative control of lentivurs treatment for lentiviruses validation of the specificity of any lentivirus target expression effects Other Ready to use lentivirus expressing specific enzymes with Enzyme different selection markers expression Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 10 of 10 www gentarget com GenTarget Inc Copyrights 2015
6. e stranded DNA cassette that to used with the standalone Cas9 expression particles Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 5 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 eli arde ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com CRISPR target sequence selection Selection of the target sequence within the gene of interest is critical to the efficacy and specificity of genetic editing with CRISPR Cas9 The crRNA segment of the gRNA will only bind to DNA targets that are immediately upstream of the proper Protospacer Adjacent Motif PAM sequence which for CRISPR Cas9 is NGG The target sequence 20bp 30bp can be in either the sense or anti sense orientation with respect to the target gene It is a good idea to create several target sequences for your gene of interest and to select sequences with minimal homology to other genes in order to find a sequence with good cleavage efficiency and minimal off target effects See the links at the bottom of the page to online bioinformatics tools to assist in selecting a gRNA sequence with minimal off target effects Online tools for target sequence selection 5 20 30 target sequence PAM NGG Note the selected sequences are in front of the NGG in genomic sequence but NGG should not be included in the synthesized gRNA http zifit partners org ZiFiT Introd
7. ell of 24 well plate where cell density is 50 to 75 After 24 72 hours no need to change medium visualize positive transduction rate by fluorescence Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 7 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 eli arde ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com microscopy For stable cell line generation pass cells into medium containing antibiotic or perform fluorescence cell sorting followed by antibiotic selection Day 0 Seed cells in complete medium at the appropriate density and incubate overnight Note at the time of transduction cells should be 50 75 confluent For example seed HeLa cells at 0 5 x 10 ml x 0 5ml in a well of a 24 well plate Day 1 s Remove the culture medium and add 0 5ml fresh warm complete medium s Thaw the pre made lentiviral stock at room temperature and add the appropriate amount of virus stock to obtain the desired MOI e Return cells to 37 C CO incubator Note Try to avoid freezing and thawing If you do not use all of the virus at one time you may re freeze the virus at 80 C for future use virus titer will decrease by 10 for each freeze thaw cycle Day 3 At 48 72hr after transduction check the transduction rate by fluorescence microscopy or calculate the exact transduction rate by flow cytometry FACS or Guava on
8. he repressor protein for the inducible expression system expression repressor Premde lentivirus for human and mouse iPS Myc NANOG iPS factors OCT4 SOX2 FLF4 factors with different fluorescent and antibitoic markers T antigen Express SV40 large T antigen with different selection markers Expression Cell Premade lentivirus for cell organelle imaging The fluorescent Organelle marker GFP RFP CFP was sub cellular localized in different imaging cell organelle for living cell imaging LacZ Express different full length B galactosidase lacZ with different expression selection markers Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 9 of 10 www gentarget com GenTarget Inc Copyrights 2015 bpn 77 21 inc 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Fax 800 3804198 Email orders gentarget com Anti miNA lentivirus Pre made lentivirus expression a specific anti miRNA cassette Fluorescent Pre made lentivirus expression a GFP RFP CFP ORF fusion ORF fusion target Pre made Premade shRNA lentivirus for knockdown a specific genes P53 shRNA LacZ Luciferase and more lentivirus microRNA Premade lentivirus expression human or mouse precursor and anti miRNA And anti miRNA lentivector and virus for human and microRNA mouse miRNA lentivirus Negative Premade negative control lentivirus with different markers control serves as
9. ly for the products containing a fluorescent marker Day 3 optional Sort transduced cells by FACS and select for antibiotic resistance A pilot experiment should be done to determine the antibiotic s kill curve for your specific cell line refer to the pertinent literature on generation of stable cell lines Note Filter wavelength settings GFP filter Ex450 490 Em525 RFP filter Ex545 Em620 Safety Precaution Gentarget lentiviral particles adapts must advanced lentiviral safety features using the third generation vectors with self inactivation SIN 3UTR and the premade lentivirus is replication incompetent However please use extra caution when using lentiviral particles Use the lentiviral particles in Bio safety II cabinet Wear glove all the time when handling Lentiviral particles Please refer CDC and NIH s guidelines for more details regarding to safety issues References 1 Ishino Y Shinagawa H Makino K Amemura M Nakata A 1987 2 Jinek M Chylinski K Fonfara I Hauer M Doudna J A Charpentier E 2012 Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 8 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 Phone 858 6788683 Fax 800 3804198 Email orders gentarget com bpn 77 21 ine 3 Hum Gene Ther 2003 14 1089 105 4 Mol Ther 2002 6 162 8 5 NIH Guidelines for Biosafety
10. mL LVP711 PBS paso Caron lentiviral aur 5x10 TFUMD LVP687 GAG h Cas9 Puro lentiviral particles particles in vivo ready 200ul 5 x 10 IFU mL LVP687 PBS Gas LETTER 200ul 5 x 107 IFU mL GAG h Cas9 Bsd lentiviral particles in vivo ready LVP688 ue 200ul 5 x 109 IFU mL h Cas9 Bsd lentiviral 7 LVP688 PB CAG 688 PBS vod ie ach 200ul 5 x 107 IFU mL LVP689 WM Caso adla 200ul 5 x 105 IFU mL LVP689 PBS CAG h Cas9 Neo lentiviral 200ul 5 x 107 IFU mL Storage lt 70 C avoid repeat freeze thaw cycles stable for gt 6 months Product Description GenTarget s lentivector system is Human Immunodeficiency Virus 1 HIV based plasmids for gene expression and knockdown The lentivectors are used to generate lentiviral particles lentivirus that can be transduced into almost all kinds of mammalian cells including stem cells primary cells and non dividing Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 2 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 eli arde ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com cells both in vivo and in vitro Lentiviral Particles stably integrate into the transduced cells genome for long term expression making it a great gene transfer agent Targeted and precise genomic gene editing technologies are the to
11. ntiviral 200ul 5 x 107 IFU mL particles in vivo ready LVP684 E cass PUPO lentiviral 200ul 5 x 108 IFU mL BM particles Lvpesa pgs EFla h Cas9 Puro lentiviral 200ul 5 x 107 IFU mL particles in vivo ready LVP685 EF1a h Cas9 Bsd lentiviral 200ul 5 x 108 IFU mL particles LvPcas pps EFta h Cas9 Bsd lentiviral 200ul 5 x 107 IFU mL Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 1 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B bpn 77 21 ine Fax San Diego CA 92126 Phone 858 6788683 800 3804198 Email orders gentarget com EFla h Cas9 Neo lentiviral particles particles in vivo ready LVP686 particles 200ul 5 x 109 IFU mL EFia h Cas9 Neo lentiviral 7 LVP686 PBS particles in vivo ready 200ul 5 x 10 IFU mL h Cas9 GFP Puro LVP708 uH Sende ura 200ul 5 x 108 IFU mL h Cas9 GFP Puro 7 LVP708 PB EFla EE lentiviral particles in vivo ready 2008 9 319 TEUER h Cas9 RFP Puro LVP709 EM ode uro 200ul 5 x 109 IFU mL h Cas9 RFP Puro 7 LELER lentiviral particles in vivo ready 2000 aE mm MM Cass GFP Bsd lentiviral sq gu 5 x 105 TFU mL j h Cas9 GFP Bsd lentiviral 7 LVP710 PBS particles in vivo ready 200ul 5 x 10 IFU mL mem EFia h Cas9 RFP Bsd lentiviral 200ul 5 x 108 IFU
12. ols for genomic correction modification and gene therapy The TALEN ZFN and CRISPR Cas are the three main genome editing technologies The lately discovered so called the third generation of gene editing technology the CRISPR Cas Clustered Regularly Interspaced Short Palindromic Repeats technology has 1 higher targeting accuracy 2 much more target sequence selection 3 much less complexity and 4 much less off target cell toxicity than the previous genome editing technologies TALEN transcription activator like effector nuclease and ZEN Zinc finger nuclease Mechanism of In CRISPR Cas systems A target sequence specific guide RNA molecule gRNA directs a cas endonuclease to the genomic DNA target sequence Then the Cas enzyme creates a double strand break at the target sequence that can be repaired by either Non Homologous End Joining NHEJ which can result in insertion or deletions InDels or correction Homology Directed Repair HDR InDels can disrupt expression of the target gene while repair by HDR which requires the presence of a repair template allows modification of the gene Cas9 the most frequently used cas endonuclease CRISPR Cas based genomic knock in out editing requires three components 1 Target specific guild RNA gRNA it comprises two segments a targeting sequence crRNA containing the target complementary RNA and an auxiliary trans activating non coding RNA sequence tracrRNA To make the gRNA
13. randed DNA cassette for sequence modification as LHA 500bp target specific left homologues arm marker insert poly A terminator RHA 500bp target specific right homologues arm e method 2 construct the donor vector clone by cloning the target specific LHA marker insert poly A terminator RHA into a donor vector 4 Add Cas9 expression lentivirus and gRNA lentivirus to target cells Note if gRNA is double stranded DNA or not lentivirus then the gRNA has to be delivered via DNA transfection such as lipid based delivery 5 If desirable for knock In apply Donor cassettes into target cells by lipid based transfection 6 select the sequence modified colonies Note If you want GenTarget to prepare the target specific gene editing reagents for you please contact GenTarget for a service quote The human codon nuclear penetrating Cas9 lentivirus are provided in two formats 1 200ul in DMEM medium with titer at 5x10 IFU ml 2 200ul concentrated virus in PBS with titer at 5x10 IFU ml For general questions about our ready to use particles please see FAQ for pre made lentiviral particles pdf on our website http www gentarget com pdf FAQ Premade Lentiviral particles pdf Transduction Protocols Note Pre made lentivirus is provided ready to use so it can be simply added into your cell culture the amount of virus to add depends on cell type For quick transduction add 50 ul of virus into each w
14. uction aspx http crispr mit edu http www e crisp org E CRISP designcrispr html http www genome engineering org CRISPR genomic editing protocol outline by using GenTarget s Cas9 lentivurs 1 select or design the 20bp target specific sequence crRNA using a online CRISPR designer tool 2 generate the gRNA that can be carried out by one of the methods listed below e method 1 or construct the gRNA transcription vector by cloning the 20nt crRNA into a gRNA vector that containing the tracrRNA already GenTarget provides services to construct your desired gRNA lentivector and ready to use gRNA lentivirus e method 2 synthesize the linear double stranded DNA cassette that transcripts the gRNA crRNA tracrRNA driven by either human U6 or H1 promoter U6 promoter crRNA tracrRNA terminator ttttttctag 369bp H1 promoter crRNA tracrRNA terminator ttttttctag 210bp Pre made Lentiviral Particles for humanized Cas9 Endonuclease Product manual Page 6 of 10 www gentarget com GenTarget Inc Copyrights 2015 7930 Arjons Drive Suite B San Diego CA 92126 eli arde ne Phone 858 6788683 Fax 800 3804198 Email orders gentarget com e method 3 By synthesize the single stranded RNA 20nt crRNA 80nt tracrRNA 100 bases crRNA 20nt tracrRNA 80nt 3 generate the Donor by the one of the methods listed below optional for knock in genomic editing s method 1 synthesize the double st
15. vide Service to construct your target specific gRNA lentivectors and their ready to use gRNA lentivirus Humanized nuclear penetrating Cas 9 expression lentivector core structure Schematic representation of lentivector for hu Cas9 expression Bsd Neo Puro GFP Puro WPRE GFP Bsd RFP Puro RFP Bsd Gentarget s Cas9 expression lentivirus has the following Key advantages e High efficient Cas9 expression delivery with markers High titer lentivirus providing more efficient Cas9 delivery in almost all cell 9 types including primary cells and non dividing cells Some Cas9 products include a fluorescent antibiotic dual marker allowing the real time check the lentivirus transduction efficiency e Different promoter selection CMV EF1a and CAG for Cas9 expression for different promoter strength in cell types e Best nuclear penetrating for Cas9 enzyme the Cas9 is expressed with an optimized proprietary Nuclear Localization Signal NLS providing the efficient cas9 delivery into the nuclear region where the gene editing occur e No need for tedious cloning work or vector construction you can simply synthesize the gRNA and donor cassette when desired and used together with the Cas9 lentivirus for the gene editing e Allow multiple gene editing at the same time no need to construct each targeting vector for different gene Instead you just select the target sequence and synthesize the gRNA each single strand RNA or doubl
Download Pdf Manuals
Related Search