(Overexpression) Product Manual. Cat#: SB-P-AV reactions. Store at -80 C upon arrival

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1 (Overexpression) Product Manual FOR RESEARCH USE ONLY Not for clinical and therapeutic use Cat#: SB-P-AV reactions Store at -80 C upon arrival AdenoONE TM - Cloning and Expression Kit FROM TRANSGENE CLONING TO ADENOVIRUS VECTOR IN ONLY 2 WEEKS CONTENTS KIT CONTENTS FOR RESEARCH USE ONLY Not for clinical and therapeutic use

2 CONTENTS KIT COMPONENTS... 2 ADDITIONAL MATERIAL REQUIRED... 2 SAFETY CONSIDERATIONS... 3 INTRODUCTION... 3 OVERVIEW... 3 VECTOR INFORMATIONS... 5 CLONING VECTOR: po6a5-cmv... 5 STEP BY STEP PROTOCOL... 6 CLONING INTO po6a5-cmv... 6 RECOMBINATION... 7 PREPARATION OF BAC DNA... 7 LINEARISATION OF BAC-DNA WITH PAC TRANSFECTION OF HEK 293 CELLS... 9 ADENOVIRUS AMPLIFICATION... 9 AMPLIFICATION STEP 1:... 9 AMPLIFICATION STEP 2: TROUBLE SHOOTING DISCLAIMER CONTACT INFORMATIONS KIT COMPONENTS Component Amount Storage Shuttle vector po6a5-cmv 10 µg - 20 C E.coli BA5-FRT electrocompetent cells 10 x 50 µl - 80 C SOC Medium 15 ml Room temperature ADDITIONAL MATERIAL REQUIRED HEK 293 cells Cell culture medium (DMEM + 10% FCS + 2 mm L-Glutamine) Cell culture Incubator (humidified, 5% CO 2 ) Laminar flow hood Cell culture centrifuge (required rcf: 4500 g) Microcentrifuge 37 C and 42 C bacterial plate incubator Electroporator and cuvettes Water bath at 37 C PIR1 chemical competent cells (Life Technologies) 37 C Shaker Thermoshaker for microfuge tubes 2

3 SAFETY CONSIDERATIONS Remember that you will be working with samples containing infectious virus. Follow the guidelines for material and organisms of biosafety level 2. INTRODUCTION Recombinant adenoviruses are widely used tools in research as well as in therapeutic applications. Due to the broad host tropism of adenoviruses they are ideal vectors for gene transfer into the majority of mammalian cells. Adenovirus are episomal vectors that do not integrate into the host genome. They allow for transient high level expression of recombinant proteins and also mediate very efficient gene knockdowns. The ability of adenoviral vectors to be purified to high titers (up to virus particles per ml) makes them ideal tools for in vivo applications. As adenovirus also possess the important attribute of stability in the bloodstream, they can be employed for gene delivery following intravenous administration. OVERVIEW The AdenoONE TM cloning system is a novel, innovative technology allowing for fast and convenient generation of recombinant adenovirus within as less as two weeks. The system is basically composed of just two major elements. Component 1: A small shuttle vector (po6a5) in which the transgene is cloned. Component 2: Electrocompetent BA5-FRT E. coli cells with a BAC vector confering restistance to chloramphenicol. On this BAC vector an Ad5 genome is contained which is devoid of the left ITR, the packaging signal, the E1 sequences and the complete E3 region. Following transformation of the shuttle vector into the electrocompentent BA5-FRT E.coli cells, Flp recombinase mediated recombination between the shuttle and the BAC vector occurs. Due to a highly sophisticated selection system only cells containing recombined BAC vector can grow and form bacterial colonies. As a result almost 100% of screened colonies contain BAC vectors with correctly recombined transgene. Therefore, the AdenoONE TM cloning system requires very limited hands on time from shuttle vector transformation to the isolation of virus particles. Virus yields and quality are absolutely comparable to those obtained with established adenoviral coning systems. 3

4 I. Transgene (TG) ITR po6a5-vector left Pac I TRANSFORMATION TG E. coli BA5-FRT po6a5-vector ITR left Pac I Pac I TG BAC SIR-BAC-Ad5 Pac I ITR right Ad5 genome II. RECOMBINATION Pac I SIR-BAC-Ad5-pO6-TG TG III. PREPARATION OF BAC DNA Pac I DIGESTION IV. VIRUS RECONSTITUTION IN 293 CELLS TG Ad5-TG Schematic overview of AdenoONE TM Cloning system STEP I: Transformation of po6a5 shuttle vector into BA5-FRT cells STEP II: Flp-mediated recombination in BA5-FRT cells STEP III: Pac 1 digestion of purified recombinant BAC-DNA STEP IV: Virus reconstitution in 293 cells 4

5 VECTOR INFORMATIONS CLONING VECTOR: po6a5-cmv (2687 bp) Multiple Cloning Site: CMV-promoter TgggaggtctatataagcagagctggtttagtgaaccgtcagatccGCTAGCACCGGT CCTGCAGG Nhe 1 Age 1 Sbf1 SV40pA GATATCACTAGTGGTACCGCGGCCGCGACtctagatcataatcagccataccacatttgtagaggtttt EcoRV Spe 1 Kpn1 SacII Not 1 Features of po6a5-cmv: Left end with L-ITR of human adenovirus serotype CMV promoter SV40 polyadenylation signal FRT site OriR6K origin of replication Kanamycin resistance gene 5

6 BAC VECTOR: SIR-BAC-Ad5 (37319 bp) SIR-BAC-Ad5 features The vector mediates resistance to Chloramphenicol (CmR) The Ad5 sequence contained in the BAC DNA is devoid of the left ITR, the packaging signal, the E1 sequences and 720 bp of the E3 region. STEP BY STEP PROTOCOL Note! It is recommended to use this product in combination with the reagents and kits specified in this manual. The protocol may need to be adapted by the customer if alternative reagents are used. CLONING INTO po6a5-cmv Day 1 Ligation Ligate your Gene of Interest (GOI) into the shuttle vector po6-a5-cmv using appropriate restriction sites. Transformation into chemical competent PIR 1 cells (available from Life Technologies) Thaw SOC medium in 37 C incubator. Once thawed store at room temperature Remove appropriate number of 20 µl aliquots of competent PIR 1 cells from the -80 C freezer.!! Using PIR1 cells is mandatory as po6a5 vectors possess the R6K origin of replication which allows plasmid propagation only in these cells!! Thaw cells on ice. Add 2 µl of ligation reaction (10-20 ng), mix carefully and place on ice for 30 min. Heat shock transformations by placing the tubes in a 42 C water bath for 40 sec. 6

7 Place tube on ice for 2 min. Add 200 µl SOC medium and incubate cells in an orbital shaker at 900 rpm for 1 h at 37 C. Plate 100 µl transformed cells onto LB plates containing 25 µg/ml Kanamycin Incubate plates at 37 C overnight. Day 2 Screening for positive colonies Day 3 The next day identify positive clones on the plates by colony PCR. Inoculate positive colonies in 10 ml LB-Kanamycin medium and shake overnight with 220 rpm at 37 C. Prepare plasmid DNA from 4 ml overnight culture using standard protocols or kits for mini-scale preparation of plasmid DNA. Expect yields in plasmid DNA between 4 and 8 µg.!!!! Purified plasmid DNA should be eluted in water as the presence of ionic compounds may lead to arching during subsequent electroporation!!!!!! Verify the presence of your cloned insert by an appropriate restriction digest. RECOMBINATION OF po6a5 SHUTTLE VECTOR INTO ADENOVIRAL BAC VECTOR For each expression construct thaw up one vial BA5-FRT electrocompetent cells on ice. Place the appropriate number of 0,2 mm electroporation cuvettes on ice which were precooled at -20 C for at least one hour. To each vial of BA5-FRT cells add 1-2 µl ( ng) of purified po6-a5 plasmid DNA and mix carefully. Transfer electrocompetent cells into a 0,2 mm electroporation cuvette and immediately electroporate at 2,5 kv, 200 Ω and 25 µf (e.g. Bio-Rad Gene Pulser, Prgm:EC2). Immediately add 1 ml SOC medium (prewarmed to 30 C) and transfer the whole content of the cuvette into a 1,5 ml microfuge tube. Shake cells at 30 C and 1000 rpm in a thermomixer for 1 h. Spin down cells at 5000 x g for 5 min, resuspend cells in 200 µl SOC medium and plate completely onto LB plates containing 25 µg/µl kanamycin and 25 µg/µl chloramphenicol. Incubate plates at 42 C for h. PREPARATION OF BAC DNA Day 4 Inoculate 100 ml LB medium supplied with 25 µg/ml chloramphenicol and 25 µg/ml kanamycin with a single colony and grow culture overnight at 37 C with shaking 7

8 (200 rpm). As AdenoONE TM recombination generates near 100% true positive clones it is usually sufficient to grow a single colony per construct. Day 5 Prepare BAC DNA using an appropriate purification kit according to the manufacturer s instructions. SIRION Biotech recommends Nucleobond PC-100 (Macherey and Nagel). As BACs are propagated in E. coli in single copy typical yields of BAC DNA are usually only between µg from a 100 ml overnight culture. LINEARISATION OF BAC-DNA WITH PAC 1 For each purified BAC DNA set up a 100 µl restriction digest as follows: SIR-BAC-Ad5-pO6-CMV (10 µg) x µl 10 x NEB 2 buffer 10 µl 10 x BSA (NEB) 10 µl Deionized water Ad 100 µl Pac I (10U/µl 1 µl Incubate at 37 C for 1,5 h Add 50 µl phenol/chloroform and vortex for 20 sec. Spin tubes in a microcentrifuge at maximum speed for 5 min. Transfer 80 µl of the aqueous upper phase into a fresh tube. Add 10 µl 3 M NaAc ph:4.5 and 200 µl EtOH. Mix with finger tips. The precipitated DNA should become visible. Incubate for 5 min at room temperature. Spin down tubes in a microcentrifuge at maximum speed for 5 min. Remove supernatant quantitatively and immediately dissolve pellet in 20 µl sterile deionized water Analyse 2-3 µl of linearized BAC DNA on a 0.8 % agarose gel. You should obtain a fragment pattern as shown below. - - Linearized Ad5 vector - - BAC portion - - doubly inserted - po6a5 vector Restriction pattern of Pac 1 digested SIR-BAC- Ad5-pO6-CMV-GOI DNAs analysed on a 0,8% agarose gel. The linearized adenoviral expression vectors migrate clearly visible above the 10 kb marker band. The excised BAC portion is represented by the 8 kb band. About 10% of BAC vectors contain double insertions of the po6a5 shuttle plasmid. In those cases the excess plasmid is excised by Pac1 and an additional band is found (4,5 kb band in lane 1). 8

9 TRANSFECTION OF HEK 293 CELLS Day 8 The day before transfection plate 2,5 x 105 HEK 293 cells per well of a 6 well plate. Per well transfect HEK 293 cells with 8 µl (ca. 3 µg) Pac 1 linearised BAC DNA using jetpei TM tranfection reagent (Polyplus transfection) according to the manufacturer s instructions. Incubate plates for 3 days under standard cell culture conditions. Harvest cells by rinsing the plate with the medium for several times until all cells are detached from the plate surface. Collect cell suspension in a sterile 15 ml tube. Centifuge for 5 min at 100 g. Remove supernant and resuspend cell pellet in 400 µl DMEM + 10% FCS + 2mM L-Glutamine. Place tube into liquid nitrogen for 3 minutes, Immediately place tube in 37 C water bath until cells have thawed completely. Repeat these steps twice. Centrifuge sample for 10 min at 4500 g in a cell culture centrifuge and transfer supernatant (= 1st rescue) into a fresh tube. Proceed immediately with next step (Adenovirus amplificaton). ADENOVIRUS AMPLIFICATION Amplification step 1: The day before adenovirus infection plate 2,5 x 105 HEK 293 cells per well of a 6 well plate. Infect each well with 200 µl 1st rescue. Incubate plates for 3-4 days under standard cell culture conditions until most cells show cytopathic effect (CPE). Days 11/12 Harvest cells by rinsing the plate with the medium for several times until all cells are detached from the plate surface. Collect cell suspension in a sterile 15 ml tube. Centifuge for 5 min at 100 g. Remove supernant and resuspend cell pellet in 400 µl DMEM + 10% FCS + 2mM L-Glutamine. Place tube into liquid nitrogene for 3 minutes. Immediately place tube in 37 C water bath until cells have thawed completely. Repeat these steps twice. Centrifuge sample for 10 min at 4500 g in a cell culture centrifuge and transfer supernatant (= 2nd rescue) into a fresh tube. Store 2nd rescue at -80 C or directly 9

10 continue with amplification step 2. Amplification step 2: The day before adenovirus infection plate 5,0 x 106 HEK 293 cells onto a 15 cm dish. Infect cells with 150 µl 2nd rescue. Incubate plates for 1-3 days under standard cell culture conditions until most cells show cytopathic effect (CPE). Days 12/14 Harvest cells by rinsing the plate with the medium for several times until all cells are detached from the plate surface. Collect cell suspension in a sterile 50 ml tube. Centifuge for 5 min at 100 g. Remove supernant and resuspend cell pellet in 400 µl DMEM + 10% FCS + 2mM L-Glutamine. Place tube into liquid nitrogene for 3 minutes. Immediately place tube in 37 C water bath until cells have thawed completely. Repeat these steps twice. Centrifuge sample for 10 min at 4500 g in a cell culture centrifuge and transfer supernatant into a fresh tube. Store adenoviral lysate at -80 C or immediately proceed with viral purification using AdenoONE TM -Purification Kit. TROUBLE SHOOTING Problem No bacterial colonies are obtained following transformation of ligation reaction of your GOI with the po6a5-cmv. No bacterial colonies are obtained following transformation of po6a5-cmv-goi into BA5- FRT cells. Cause Cells other than PIR1 were used for transformation: Use PIR1 cells po6a5-cmv-goi preparation was not free of salts Electroporation cuvettes were not precooled Amount of po6a5 DNA used was either too low or to high Plates were incubated at 37 C instead of 42 C Virus rescue failed Either substantially less or more than 3 µg Pac 1 digested BACl DNA was used for transfection of 293 cells. Confluency of 293 cells was too high. 10

11 DISCLAIMER This product is sold for research and development purposes only and is not to be incorporated into products for resale without the permission from SIRION BIOTECH GmbH and may not be given to third parties. CONTACT INFORMATIONS SIRION BIOTECH GmbH Am Klopferspitz Martinsried Germany Phone: info@sirion-biotech.de 11