Adeno-1 Expression System
Contents
1. Ampr transformants by plating the transformation mixture on a LB agar Amp plate 100 ug ml ampicillin Incubate overnight at 37 C Pick up 10 colonies and inoculate into 100 ul LB Amp medium in a sterile Eppendorf tube in the early morning After 5 6 hours of incubation screen for positive clones using PCR with Adeno screening primer mix using the protocol in Appendix B When you have identified a bacterial clone carrying the desired recombinant inoculate 100 500 ml of LB Amp medium with all the bacteria 100 ul from the 1 5 ml Eppendorf tube Incubate the culture at 37 C overnight Purify the plasmid using our Column Pure Plasmid Maxi Prep Kit Cat No D205 or the Qiagen kits Expected yield 30 50 ug plasmid DNA 100 ml culture Note pAdeno is a large plasmid gt 30 kb that is susceptible to damage and rearrangement in E coli For best results always use fresh log phase cultures for purification of recombinant pAdeno DNA Do not store your culture at room temperature 4 C or on ice for long periods i e gt 24 hours before starting purification 3 2 3 Analyze putative recombinant Adenoviral DNA by PCR or restriction digestion PI Sce I and I Ceu I Restriction Analysis a Set up 30ul PI Sce I I Ceu I double digests by combining the reagents in Table 4 in a sterile 1 5 ml microcentrifuge tube Mix well and spin briefly to collect contents Incubate at 37 C for exactly 3 hours Verify the insert by elec2. pAdeno 1 I Ceu I PI Sce I ee O Pac I Your gene expression cassette from our pShuttle vectors Ligation and Swa I digestion x Transformation and recombinant adenoviral DNA Purification Y Pac I digestion to linearize v Transfect low passage HEK 293 cells Collect recombinant adenovirus 3 Experimental Procedure 3 1 Cloning your gene of interest into the pShuttle 3 1 1 Subclone your gene of interest into the pShuttle using any of the unique restriction sites located in the MCS region Ceu Carr Af If Hind ff Asp718 I Kpn I g lt cT AGC GTT TAA ACT iij oy TGG CoA GCT eee ATC CAC TAG TCC AGT GIG GTG GAA TTC Pst I EcorR V Bst XI Nat I Xho I pShuttle TGC AGA TAT GEA GCA CAG TGG CGG CCG CTC GAG ii Aba I Apa Pme I TCT AGA GGG CCC GTT ma AC 3 MCS a Pl Sce i843 X Ceu I ary Nhe I Fme f A I Aba T Aho I 5 GCT AGC GTT TAA ACG GGC CCT CTA GAC TCG Eco RI CMV nec sa zM CAC TGT GCT GGA TAT CTG CAG AAT pShuttle Aspi 18 J 4817 bp TCC ACC ACA CTG GAC TAG TGG ATC CGA GCT CGG nen Hind IT ARH j TAC CAA GCT TAA GTT TAA AC 3 21530 Pl sce 1843 ORI To 3 1 2 PI Sce I I Ceu I digestion of recombinant pshuttle and adeno DNA a Prepare a 30ul PI Sce I I Ceu I double digest of your pShuttle and adeno DNA b Combine the reagents shown in Table 2 in a sterile 1 5 ml microcentrifuge tube c Mix well and spin briefly to collec
3. packaging cells 1 Plate 293 cells at 70 confluency in a 6 well plate 12 24 hrs before transfection 2 Incubate the plate s at 37 C in a humidified atmosphere maintained at 5 CO 3 Transfect each 6 well culture plate with 10 ul of Pac I digested recombinant Adenoviral DNA Use Celfectin Cat No G061 to transfer DNA into 293 cells 4 Subculture cells into a T75 flask using trypsin 3 5 days after transfection 5 Check periodically for cytopathic effect CPE Note It normally takes 7 12 days to see CPE 6 When gt 90 of the cells have detached from the plate prepare viral stock by following steps 7 10 Name this stock Primary Amplification and store at 20 C Primary Amplification Stock is suitable for infecting target cells We suggest you evaluate the function of this viral stock before preparing High Titer Stock The presence of your recombinant construct can be verified by PCR or Western blotting 7 Centrifuge the suspension at 1 500 x g for 5 min at room temperature 8 Re suspend the pellet in 500 ul sterile PBS 9 Lyse cells with three consecutive freeze thaw cycles Freeze cells in dry ice ethanol bath thaw cells by placing the tube in a 37 C water bath Do not allow suspension to reach 37 C 10 After the third cycle briefly centrifuge to pellet debris Transfer the lysate to a clean sterile centrifuge tube and store the lysate at 20 C 11 Determine the adenoviral titer The Adenove
4. Wa Capital Biosciences innovative solutions for life sciences Adeno 1 Expression System Enables efficient construction of recombinant adenoviruses Store kit at 20 C Store Ad competent cell at 70 C User Manual Version 2 January 2007 www capitalbiosciences com 1 Introduction Application The Adeno 1 Expression System provides an efficient method for constructing recombinant adenoviruses Our procedure uses in vitro ligation to subclone your gene of interest into a replication incompetent E1 E3 human adenoviral type 5 Ad5 genome This approach enables you to produce recombinant adenoviruses quickly less than 3 weeks and reliably Our pShuttle vectors have very flexible cloning sites which allow easy subcloning manipulation 2 Kit Contents Table 1 Kit Contents Component Adeno DNA pShuttle vector pShuttle vector pShuttle Laz control vector Enzyme pack PI Sce I and I Ceul including buffer Recombinant adenovirus PCR screening set 293 cells Ad competent cells Glycogen 20mg ml Kit Verizon ee v1 5 v1 6 Sug vA Sug V Sug vA Sug vA 2x50ul v Z 2 2x100ul v 2 1x10e6 y 10x50ul 7 100ul P Store Ad competent cells at 70 C and all other components at 20 C Spin briefly to recover contents Avoid repeated freeze thaw cycles I Ceul Swal PlSce I Pacl wha pUC ori pAdeno E 33 5 kb la PacI I Ceu I PI Sce I
5. ctor Rapid Titer Kit Cat VSA 0024 enables you to determine the adenoviral titer by using an anti Hexon antibody cell staining assay 3 4 Infecting target cells If the viruses are to be used in in vitro cell cultures Double CsCl purification is not required as the viral supernatant will provide 100 gene transduction efficiency in most human cell lines For in vivo studies i e animal studies purification is essential to remove defective particles cell debris and small amounts of media components since these contaminants can induce significant immune responses In addition CsCl purification will concentrate the virus to a level suitable for in vivo injections Appendix A Phenol Chloroform Isoamyl Alcohol Extraction 1 13 Top up the sample to 100ul with TE buffer pH 8 0 then add 100ul phenol chloroform isoamyl alcohol 25 24 1 Vortex thoroughly Spin the tube in a microcentrifuge at 14 000 rpm for 2 min at room temperature to separate phases Carefully transfer the top aqueous layer to a clean 1 5 ml tube Add 400 ul 95 ethanol 25 ul 10 M NH4OAc or 1 10 volume of 3 M NaOAc and 1 ul glycogen 20 mg ml Vortex thoroughly Spin the tube at 14 000 rpm for 5 min at room temperature Remove and discard the supernatant Carefully overlay the pellet with 300 ul 70 ethanol Spin in a microcentrifuge at 14 000 rpm for 2 min at room temperature Carefully aspirate off the supernatant Air dry the pellet
6. for approximately 3 5 min at room temperature to evaporate residual ethanol Dissolve the DNA in 10 ul sterile 1X TE Buffer pH 8 0 for down stream applications or store at 20 C until use Appendix B Identification of Recombinant Adenoviral DNA by PCR 1 2 3 Set up the reaction as following using 2x PCR Taq MasterMix Cat No G013 or your own Taq DNA polymerase Template bacterial culture or purified DNA 1 ul Primers mix of both forward and reverse 2 ul ddH O 22 ul 2x PCR Taq MasterMix 25 pl Total 50 pl Perform PCR as following Denaturation 95 C 4 min PCR Cycles 30 35 94 C 30 sec pT 30 sec TC 1 min Final elongation 72 5 min After PCR perform DNA agarose gel electrophoresis Positive band is at 350bp Make sure to include a negative control for each assay
7. t liquid Incubate at 37 C for exactly 3 hours e Perform phenol chloroform isoamyl alcohol 25 24 1 extraction by using the protocol in Appendix A Table 2 PI Sce I I Ceu I Double Digest of pShuttle and pAdeno DNA Reagent Volume pshuttle Plasmid or pAdeno DNA 2 3ug 10X Double Digestion Buffer 3ul PI Sce I Restriction Enzyme 1 unit ul 2ul I Ceu I Restriction Enzyme 1 unit ul 2ul 10X BSA 3ul Sterile H2O x ul Total Volume 30ul 3 2 Produce recombinant adenoviral DNA 3 2 1 Subclone your expression cassette into the Adeno genome a nN Combine the reagents shown in Table 3 in a sterile 1 5 ml microcentrifuge tube in the order as shown Gently mix then spin briefly in the microcentrifuge Incubate at room temperature for 1 2 hours To the ligation product add the following 1 5 ul 10X Swa I digestion Buffer 1 5 ul 10X BSA 1 0 ul Swa I enzyme 1 0 ul ddH2O Incubate at room temperature for 1 2 hours Perform phenol chloroform isoamyl alcohol 25 24 1 extraction by using the protocol in Appendix A Table 3 Ligation of pShuttle to Adenovector Reagent Volume PI Sce I I Ceu I digested vector 4ul PI Sce I I Ceu I digested pShuttle 3ul 5X DNA Ligation Buffer 2ul DNA Ligase lul Total Volume 10ul 3 2 2 Transform E coli with ligation products a Transform electro or chemically competent DHsa or our Ad competent Cat VSA 0002 cells with the Swal digestion product Select for ampicillin resistant
8. trophoresis on a 0 8 1 agarose SafeView Cat No DG108 gel Important Since I Ceu I and PI Sce I tend to remain bound to DNA use a gel loading buffer that contains SDS final concentration after combining with sample 0 1 heat to 65 C for 10 minutes before loading Table 4 Restriction Analysis of Recombinant Adenoviral DNA Reagent Volume Adenoviral DNA 3ug 10X Digestion Buffer 3ul 10X BSA 3ul PI Sce I 1 unit ul 2ul I Ceu I 1 unit ul 2ul ddH2O0 x ul Total Volume 30ul PCR Analysis You can also screen pAdeno DNA for the presence of pShuttle derived expression cassettes by using PCR with Adeno forward and reverse PCR primers using the protocol in Appendix B These primers specifically amplify a 350 bp sequence that spans the cloning site in pAdeno Only recombinant pAdenoviral templates are amplified since non recombinants lack the pShuttle sequence needed for annealing with the reverse primer 3 2 4 Preparing Recombinant Adenoviral DNA for Transfection a Ina sterile 1 5 ml microcentrifuge tube combine the reagents in Table 5 b Mix contents and spin the tube briefly c Incubate at 37 C for 2 hours d Perform phenol extractions by using the protocol in Appendix A Table 5 Pac I Digestion of Recombinant Adenoviral DNA Reagent Volume Recombinant Adenoviral DNA 3ug 10X Pac I Digestion Buffer 4ul 10X BSA 4ul Pac I 10 units ul 2ul ddH20 x ul Total Volume 40ul 3 3 Generate the recombinant adenovirus in
Download Pdf Manuals
Related Search