The ViraPower™ Adenoviral Expression System, continued
Contents
1. e The entire El region is deleted in the pad CMV V5 DEST or pAd PL DEST expression vectors Expression of the El proteins is required for the expression of the other viral genes e g late genes and thus viral replication only occurs in cells that express El Graham et al 1977 Kozarsky and Wilson 1993 Krougliak and Graham 1995 e Adenovirus produced from the pAd CMV V5 DEST or pAd PL DEST expression vectors is replication incompetent in any mammalian cells that do not express the Ela and Elb proteins Graham et al 1977 Kozarsky and Wilson 1993 Krougliak and Graham 1995 e Adenovirus does not integrate into the host genome upon transduction Because the virus is replication incompetent the presence of the viral genome is transient and will eventually be diluted out as cell division occurs Despite the presence of the safety features discussed above the adenovirus produced with this System can still pose some biohazardous risk since it can transduce primary human cells For this reason we highly recommend that you treat adenoviral stocks generated using this System as Biosafety Level 2 BL 2 organisms and strictly follow all published guidelines for BL 2 Furthermore exercise extra caution when creating adenovirus carrying potential harmful or toxic genes e g activated oncogenes or when producing large scale preparations of virus see the next page For more information about the BL 2 guidelines and adenovirus handling2. refer to the document Biosafety in Microbiological and Biomedical Laboratories 4 Edition published by the Centers for Disease Control CDC This document may be downloaded from the Web at the following address http www cdc gov od ohs biosfty bmbl4 bmbl4toc htm continued on next page Biosafety Features of the System continued Additional Cautions When Producing Large Scale Prepara tions of Virus The genomic copy of El in all 293 cell lines contains homologous regions of overlap with the pAd CMV V5 DEST and pAd PL DEST vectors In rare instances itis possible for homologous recombination to occur between the El genomic region in 293 cells and the viral DNA causing the gene of interest to be replaced with the El region and resulting in generation of a wild type replication competent adenovirus RCA Lochmuller et al 1994 This event is most likely to occur during large scale preparation or amplification of virus and the growth advantages of the RCA allow it to quickly overtake cultures of recombinant adenovirus To reduce the likelihood of propagating RCA contaminated adenoviral stocks e Use caution when handling all viral preparations and treat as BL 2 see the previous page and page 14 for more details e Perform routine screening for the presence of wild type RCA contamination after large scale viral preparations Suitable methods to screen for RCA contamination include PCR screening Zha
3. 3 Store at 4 C for up to 6 months 25 Technical Service Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_service invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com Material Data Safety Sheets MSDSs Limited Warranty 26 MSDSs are available on our Web site at www invitrogen com On the home page click on Technical Resources and follow instructions on the page to download the MSDS for your product Invitrogen is committed to providing our cust
4. Adenoviral Vectors Hum Gene Ther 7 215 222 Fan X Brun A Segren S Jacobsen S E and Karlsson S 2000 Efficient Adenoviral Vector Transduction of Human Hematopoietic SCID Repopulating and Long Term Culture Initiating Cells Hum Gene Ther 11 1313 1327 Graham F L Smiley J Russell W C and Nairn R 1977 Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5 J Gen Virol 36 59 74 Hitt M M Parks R J and Graham F L 1999 Structure and Genetic Organization of Adenovirus Vectors In The Development of Human Gene Therapy T Friedmann ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press pp 61 86 Kozarsky K F and Wilson J M 1993 Gene Therapy Adenovirus Vectors Curr Opin Genet Dev 3 499 503 Krougliak V and Graham F L 1995 Development of Cell Lines Capable of Complementing El F4 and Protein IX Defective Adenovirus Type 5 Mutants Hum Gene Ther 6 1575 1586 31 References Lochmuller H Jani A Huard J Prescott S Simoneau M Massie B Karpati G and Acsadi G 1994 Emergence of Early Region 1 Containing Replication Competent Adenovirus in Stocks of Replication Defective Adenovirus Recombinants Delta E1 Delta E3 During Multiple Passages in 293 Cells Hum Gene Ther 5 1485 1491 Navarro V Millecamps S Geoffroy M C Robert J J Valin A Mallet J and Salle G L G L 1999 E
5. be suitable for use for up to one year After long term storage we recommend re titering your viral stocks before use 13 Amplifying Your Adenoviral Stock Introduction Note Materials Needed 14 Once you have created a crude viral stock you can use this stock to infect 293A cells to generate a higher titer viral stock i e amplify the virus The titer of the initial viral stock obtained from transfecting 293A cells generally ranges from 1 x 10 to 1x 10 plaque forming units pfu ml Amplification allows production of a viral stock with a titer ranging from 1 x 10 to 1x 10 pfu ml and is generally recommended Guidelines and protocols are provided in this section to amplify the recombinant adenovirus using 293A cells plated in a 10 cm dish Larger scale amplification is possible see page 16 Note Other 293 cell lines or cell lines expressing the El proteins are suitable Remember that you will be working with infectious virus Follow the recommended Federal guidelines for working with BL 2 organisms e Perform all manipulations within a certified biosafety cabinet e Treat media containing virus with bleach e Treat used pipets pipette tips and other tissue culture supplies with bleach or dispose of as biohazardous waste e Wear gloves a laboratory coat and safety glasses or goggles when handling viral stocks and media containing virus We have not observed wild type RCA contamination in small
6. cells by squirting cells off the plate with a 10 ml tissue culture pipette Transfer cells and media to a sterile 15 ml capped tube 5 Place the tube containing harvested cells and at 80 C for 30 minutes Remove tube and place in a 37 C water bath for 15 minutes to thaw Repeat the freezing and thawing steps twice 6 Centrifuge the cell lysate in a table top centrifuge at 3000 rpm for 15 minutes at room temperature to pellet the cell debris 7 Transfer the supernatant containing viral particles to cryovials in 1 ml aliquots Store the viral stocks at 80 C For long term storage store as described on page 13 Proceed to Titering Your Adenoviral Stock next section continued on next page 15 Amplifying Your Adenoviral Stock continued Scale Up 16 The amplification procedure is easily scalable to any size tissue culture dish or roller bottle If you wish to scale up the amplification remember that you will need to increase the number of cells and amount of crude viral stock and medium used in proportion to the difference in surface area of the culture vessel Important Reminder Remember to screen for the presence of wild type RCA contamination in your amplified stock Refer to published references for suitable screening protocols Dion et al 1996 Zhang et al 1995 Titering Your Adenoviral Stock Introduction Experimental Outline Factors Affecting Viral Titer Before proceeding to transduce
7. in a level tissue culture hood at room temperature for 15 minutes or until the agarose overlay solidifies Return the plate to a 37 C humidified CO incubator 2 days following the initial overlay Day 5 gently overlay the cells with an additional 1 ml of agarose overlay solution per well Prepare the agarose overlay solution as described in Step 5 Allow the agarose overlay to solidify before returning the plate to a 37 C humidified CO incubator Monitor the plates until plaques are visible generally 8 12 days post infection Day 10 14 For each well gently layer the 5 mg ml MTT solution 1 10 the volume of the agarose overlay on top of the solidified agar to stain Make sure the MTT solution is evenly distributed over the entire surface of the well Example If each well contains 3 ml of agarose overlay use 300 ul of 5 mg ml MTT Incubate plates for 3 hours at 37 C Count the plaques and determine the titer of your adenoviral stock continued on next page 19 Titering Your Adenoviral Stock continued What You Should See Concentrating Virus 20 When titering pAd CMV V5 DEST or pAd PL DEST adenoviral stocks using 293A cells we generally obtain titers ranging from 1 x 10 to 1x 10 pfu ml Adenoviral constructs with titers in this range are generally suitable for use in most applications Note If the titer of your adenoviral stock is less than 1 x 107 pfu ml we recommend producing a new adenoviral s
8. including the nature of your mammalian cell line e g non dividing vs dividing cell type see Note on the next page its transduction efficiency your application of interest and the nature of your gene of interest If you are transducing your adenoviral construct into the mammalian cell line of choice for the first time we recommend using a range of MOIs e g 0 0 5 1 2 5 10 20 50 to determine the MOI required to obtain optimal expression of your recombinant protein for your particular application continued on next page 21 Transduction and Analysis continued Note Positive Control Important Transduction Procedure Detecting Recombinant Protein 22 In general we have found that 80 90 of the cells in an actively dividing cell line e g HT1080 express a target gene when transduced at an MOI of 1 Other cell types including non dividing cells may transduce adenoviral constructs less efficiently If you are transducing your adenoviral construct into a non dividing cell type you may need to increase the MOI to achieve optimal expression levels for your recombinant protein If you have generated the pAd CMV V5 GW lacZ control adenoviral construct we recommend using the adenoviral stock to help you determine the optimal MOI for your particular cell line and application Once you have transduced the Ad CMV V5 GW lacZ adenovirus into your mammalian cell line of choice the gene encoding P galactosidas
9. scale i e 3 x 10 293A cells plated in a 10 cm dish adenoviral amplification using the protocol on page 15 However if you plan to perform large scale amplification of virus we recommend screening for wild type RCA contamination Note that even in large scale preparations contamination of adenoviral stocks with wild type RCA is a rare event For more information see page 6 You should have the following materials on hand before beginning e Crude adenoviral stock of your pAd DEST construct from Preparing a Crude Viral Lysate Step 3 page 13 Note If you have produced an adenoviral stock of the pAd CMV V5 GW lacZ construct we recommend amplifying this viral stock as well e 293A cells cultured in the appropriate medium see the 293A Cell Line manual for details e Sterile 10 cm tissue culture plates e Sterile tissue culture supplies e 15 mlsterile capped conical tubes e Table top centrifuge e Water bath set to 37 C e Cryovials continued on next page Amplifying Your Adenoviral Stock continued Infection To amplify the adenoviral stock we typically infect 293A cells using the following Conditions conditions Condition Amount Tissue culture plate size 10 cm one per adenoviral construct Number of 293A cells to infect 3 x 10 cells Amount of crude adenoviral stock to use 100 ul see Note below We generally infect a 10 cm plate of 293A cells with 100 ul of untitered cru
10. some potential problems and possible solutions that may help you troubleshoot your transduction and expression experiment Problem Reason Solution No expression Viral stocks stored incorrectly Aliquot and store stocks at 80 C Do not freeze thaw more than 10 times Gene of interest contains a Pac 1 site Perform mutagenesis to change or remove the Pac I site Poor expression Poor transduction efficiency e Mammalian cells not healthy e Non dividing cell type used e Make sure that your cells are healthy before transduction Transduce your adenoviral construct into cells using a higher MOI MOI too low Transduce your adenoviral construct into cells using a higher MOI Low viral titer Amplify the adenoviral stock using the procedure on page 15 Adenoviral stock contaminated with RCA e Screen for RCA contamination Dion et al 1996 Zhang et al 1995 e Prepare a new adenoviral stock or plaque purify to isolate recombinant adenovirus Cells harvested too soon after transduction Do not harvest cells until at least 24 hours after transduction Cells harvested too long after transduction For actively dividing cells assay for maximal levels of recombinant protein expression within 5 days of transduction Gene of interest is toxic to cells Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended Per
11. tube containing harvested cells and media from Transfection Procedure Step 9 page 11 at 80 C for 30 minutes Remove tube and place in a 37 C water bath for 15 minutes to thaw Repeat the freezing and thawing steps twice Note Do not incubate samples at 37 C for longer than 15 minutes 2 Centrifuge the cell lysate in a table top centrifuge at 3000 rpm for 15 minutes at room temperature to pellet the cell debris 3 Transfer the supernatant containing viral particles to cryovials in 1 ml aliquots Store the viral stocks at 80 C What to Do Next Once you have prepared a crude viral stock you may e Amplify the viral stock by infecting 293A cells see the next section for details This procedure is recommended to obtain the highest viral titers and optimal results in your transduction studies e Determine the titer see pages 17 20 for instructions e Use this viral stock to transduce your mammalian cells of interest to verify the functionality of your adenoviral construct in preliminary expression experiments see pages 21 22 for more information Long Term Place viral stocks at 80 C for long term storage Because adenovirus is non Storage enveloped viral stocks remain relatively stable and some freezing and thawing of the viral stocks is acceptable We do not recommend freezing and thawing viral stocks more than 10 times as loss of viral titer can occur When stored properly viral stocks of an appropriate titer should
12. 5 062 Phosphate Buffered Saline PBS pH 7 4 500 ml 10010 023 1L 10010 031 S N A P MidiPrep Kit 20 reactions K1910 01 Overview Introduction Advantages of the ViraPower Adenoviral System Introduction The ViraPower Adenoviral Expression System allows creation of a replication incompetent adenovirus that can be used to deliver and transiently express your gene of interest in either dividing or non dividing mammalian cells The major components of the ViraPower Adenoviral Expression System include e A choice of Gateway adapted adenoviral vectors that allow highly efficient generation of a recombinant adenovirus containing the gene of interest under the control of the human cytomegalovirus CMV immediate early enhancer promoter pAd CMV V5 DEST or a promoter of choice pAd PL DEST e An optimized cell line 293A which allows production and subsequent titering of the recombinant adenovirus e A control expression plasmid containing the lacZ gene which when packaged into virions and transduced into a mammalian cell line expresses B galacto sidase For more information about the adenoviral vectors the corresponding positive control vector containing the lacZ gene and Gateway Technology refer to the pAd CMV V5 DEST and pAd PL DEST Gateway Vector manual This manual is supplied with each ViraPower Adenoviral Expression Kit and may also be downloaded from our Web site www invitroge
13. Replenish culture medium and allow infections to proceed until approximately 80 CPE is observed typically 10 13 days post transfection Harvest adenovirus containing cells by squirting cells off the plate with a 10 ml tissue culture pipette Transfer cells and media to a sterile 15 ml capped tube Proceed to Preparing a Crude Viral Lysate page 13 continued on next page 11 Producing Adenovirus in 293A Cells continued Example of CPE In this example Pac I digested pAd CMV V5 GW lacZ plasmid was transfected into 293A cells using the recommended protocol on the previous page The photographs show transfected cells as they undergo CPE Day 4 6 post transfection At this early stage cells producing adenovirus first appear as patches of rounding dying cells Day 6 8 post transfection As the infection proceeds cells containing viral particles lyse and infect neighboring cells A plaque begins to form Day 8 10 post transfection At this late stage infected neighboring cells lyse forming a plaque that is clearly visible continued on next page 12 Producing Adenovirus in 293A Cells continued Preparing a Crude After you have harvested adenovirus containing cells and media you will use Viral Lysate several freeze thaw cycles followed by centrifugation to prepare a crude viral lysate The freeze thaw cycles cause the cells to lyse and allow release of intracellular viral particles 1 Place the
14. The vector also contains the elements required to allow packaging of the expression construct into virions e g 5 and 3 ITRs encapsidation signal adenoviral late genes For more information about the pAd DEST expression vectors refer to the pAd CMV V5 DEST and pAd PL DEST Gateway Vector manual e The second major component of the System is an optimized 293A Cell Line that will be used to facilitate initial production amplification and titering of replication incompetent adenovirus The 293A cells contain a stably integrated copy of El that supplies the E1 proteins Ela and Elb in trans required to generate adenovirus For more information about the 293A Cell Line refer to the 293A Cell Line manual You will transfect the pAd DEST vector containing your gene of interest into 293A cells to produce a replication incompetent adenovirus You will next use the crude adenoviral stock to infect 293A cells to produce an amplified adenoviral stock Once the adenoviral stock is amplified and titered this high titer stock can be used to transduce the recombinant adenovirus into the mammalian cell line of choice for expression of the recombinant protein of interest How Adenovirus Adenovirus enters target cells by binding to the Coxsackie Adenovirus Receptor Works CAR Bergelson et al 1997 After binding to the CAR the adenovirus is internalized via integrin mediated endocytosis Russell 2000 followed by active transport to the
15. clusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Purchaser Notification Introduction Limited Use Label License No 19 Cloning Technology Products Use of the ViraPower Adenoviral Expression Kits is covered under a number of different licenses including those detailed below This product and its use is the subject of one or more of U S Patent Nos 5 888 732 6 143 557 6 171 861 6 270 969 6 277 608 and 6 720 140 and or other pending U S and foreign patent applications owned by Invitrogen Corporation The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications or to use this product with any recombination sites other than those purchased from Invitrogen Corporation or its authorized distributor The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by the use of Clonase purchased from Invitrogen Corporation or its authorized distributors The buyer cannot modify the recombination sequence s contained in this product for any purpose T
16. de viral Note stock Assuming a viral titer of 1x 10 to 1x 10 pfu ml this generally allows us to harvest the desired number adenovirus containing cells 2 3 days after infection You may vary the volume of crude viral stock used to infect cells if desired We have used up to 1 ml of crude viral stock If you have determined the titer of your crude viral stock we recommend infecting 293A cells at a multiplicity of infection MOI 3 to 5 Amplification Follow the procedure below to amplify your adenoviral stock using 293A cells Procedure Make sure that your 293A cells are healthy at the time of plating 1 The day before infection trypsinize and count the 293A cells plating them at 3 x 10 cells per 10 cm plate Plate cells in 10 ml of normal growth medium containing serum 2 On the day of infection verify that the cells are at 80 90 confluency before proceeding Add the desired amount of crude adenoviral stock e g 100 ul to the cells Swirl the plate gently to mix 3 Incubate the cells at 37 C in a CO incubator and allow infection to proceed until 80 90 of the cells have rounded up and are floating or lightly attached to the tissue culture dish typically 2 3 days post infection This indicates that cells are loaded with adenoviral particles Note If you have used less than 100 ul of crude viral stock or a lower titer stock for infection you may need to perform a longer incubation 4 Harvest adenovirus containing
17. e any method of choice to prepare purified plasmid DNA Remember that plasmid DNA for transfection into eukaryotic cells must be clean and free from phenol and sodium chloride as contaminants may kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the S N A P MidiPrep Kit Catalog no K1910 01 or CsCl gradient centrifugation The human 293A Cell Line is included with the ViraPower Adenoviral Expression kits to facilitate adenovirus production from the E1 deleted pAd DEST vectors The 293A Cell Line a subclone of the 293 cell line supplies the E1 proteins in trans that are required for expression of adenoviral late genes and thus viral replication The cell line exhibits a flattened morphology enabling easier visualization of plaques For more information about how to culture and maintain 293A cells refer to the 293A Cell Line manual Note Any 293 derived cell line or other cell line that expresses the E1 proteins may be used to produce adenovirus Producing Adenovirus in 293A Cells Introduction Preparing the Expression Clone for Use Materials Needed Once you have created a pAd DEST expression clone you will transfect the expression clone into 293A cells to produce an adenoviral stock The following section provides protocols and instructions to generate an adenoviral stock Before you can transfect your pAd DEST expressio
18. e will be constitutively expressed and can be easily assayed refer to the pad CMV V5 DEST and pAd PL DEST Gateway Vector manual for details Remember that viral supernatants are generated by lysing cells containing virus into spent media harvested from the 293A producer cells Spent media lacks nutrients and may contain some toxic waste products If you are using a large volume of viral supernatant to transduce your mammalian cell line e g 1 ml of viral supernatant per well in a 6 well plate note that growth characteristics or morphology of the target cells may be affected during transduction These effects are generally alleviated after transduction when the media is replaced with fresh complete media Follow the procedure below to transduce the mammalian cell line of choice with your adenoviral construct 1 Plate your mammalian cells of choice in complete media as appropriate for your application 2 On the day of transduction Day 1 thaw your adenoviral stock and dilute if necessary the appropriate amount of virus at a suitable MOI into fresh complete medium Do not vortex 3 Remove the culture medium from the cells Mix the medium containing virus gently by pipetting and add to the cells Swirl the plate gently to disperse the medium Incubate at 37 C overnight 4 The following day Day 2 remove the medium containing virus and replace with fresh complete culture medium 5 Harvest the cells if needed on
19. ectious viral progeny are generated Mammalian cell lines that do not express El are transduced In this case you are using adenovirus as a gene delivery vehicle The ViraPower Adenoviral Expression System is suitable for in vivo gene delivery applications Many groups have successfully used adenoviral vectors to express a target gene in a multitude of tissues including skeletal muscle lung heart and brain For more information about target genes that have been successfully expressed in vivo using adenoviral based vectors refer to the published reviews Russell 2000 Wang and Huang 2000 Wivel 1999 Biosafety Features of the System Introduction Information for European Customers Biosafety Features of the ViraPower Adenoviral System Biosafety Level 2 The ViraPower Adenoviral Expression System is a second generation system based on adenoviral vectors developed by Bett et al 1994 This second generation adenoviral system includes a number of safety features designed to enhance its biosafety These safety features are discussed below The 293A Cell Line is genetically modified and carries adenovirus type 5 sequences As a condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms The ViraPower Adenoviral Expression System includes the following safety features
20. ector manual for a description of the criteria used to qualify the adenoviral vectors Each lot of cells is tested for cell growth and viability post recovery from cryopreservation The flat morphology is verified by visual inspection Master Cell Banks are screened for viruses mycoplasma and sterility Using the reagents provided in the kit the pAd CMV V5 GW lacZ plasmid is transfected into 293A cells using the protocol on page 11 Cells are harvested 10 days post transfection and a crude viral lysate is prepared using the protocol on page 13 The crude viral lysate is used to infect 293A cells cells are harvested 3 days post infection and viral supernatant is prepared and titered The pAd CMV V5 GW lacZ adenoviral construct must demonstrate a titer of greater than 1 x 10 pfu ml and must express functional galactosidase when transduced into HT1080 cells 29 30 References Bergelson J M Cunningham J A Droguett G Kurt Jones E A Krithivas A Hong J S Horwitz M S Crowell R L and Finberg R W 1997 Isolation of a Common Receptor for Coxsackie B Viruses and Adenoviruses 2 and 5 Science 275 1320 1323 Bett A J Haddara W Prevec L and Graham F L 1994 An Efficient and Flexible System for Construction of Adenovirus Vectors with Insertions or Deletions in Early Regions 1 and 3 Proc Natl Acad Sci USA 91 8802 8806 Chen H H Mack L M Choi S Y Ontell M Koc
21. ector manual is supplied with each ViraPower Adenoviral Expression Kit and may also be downloaded from our Web site www invitrogen com or requested from Technical Service page 26 Each ViraPower Adenoviral Expression Kit includes the 293A Cell Line to facilitate production of adenovirus Refer to the 293A Cell Line manual for detailed information about the amount of cells provided and instructions on how to culture and maintain the cell line The 293A Cell Line manual is supplied with each ViraPower Adenoviral Expression Kit and may also be downloaded from our Web site www invitrogen com or requested from Technical Service see page 26 Accessory Products Introduction Additional Products vi The products listed in this section may be used with the ViraPower Adenoviral Expression Kits For more information refer to our Web site www invitrogen com or call Technical Service see page 26 The reagents supplied in the ViraPower Adenoviral Expression Kits as well as other products suitable for use with the kits are available separately from Invitrogen Ordering information is provided below Item Quantity Catalog no pAd CMV V5 DEST Gateway Vector 6 ug V493 20 pAd PL DEST Gateway Vector 6 ug V494 20 293A Cell Line 3x 10 cells frozen R705 07 Lipofectamine 2000 0 75 ml 11668 027 1 5 ml 11668 019 Opti MEM I Reduced Serum Medium 100 ml 31985 062 500 ml 3198
22. ed an adenoviral stock of the pAd CMV V5 GW lacZ positive expression control we recommend titering this stock as well 1 The day before infection Day 1 trypsinize and count the cells plating them such that they will be 80 90 confluent at the time of infection Incubate cells at 37 C overnight Example When using 293A cells we generally plate 1 x 10 cells per well in a 6 well plate On the day of infection Day 2 thaw your adenoviral stock and prepare 10 fold serial dilutions ranging from 10 to 10 For each dilution dilute the adenoviral construct into complete culture medium to a final volume of 1 ml Do not vortex Remove the culture medium from the cells Mix each dilution gently by inversion and add to one well of cells total volume 1 ml Swirl the plate gently to disperse the media Incubate at 37 C overnight The following day Day 3 remove the media containing virus and gently overlay the cells with 2 ml of agarose overlay solution per well Prepare the agarose overlay solution enough to overlay one 6 well plate at a time as described below e For one 6 well plate 2 ml overlay per well gently mix 12 ml of pre warmed at 37 C plaquing media and 1 2 ml of pre warmed at 65 C 4 agarose Avoid formation of bubbles e Apply the overlay to the cells by gently pipetting the overlay down the side of each aspirated well Work quickly to prevent premature solidification e Place the 6 well plate
23. expression over time generally to background levels within 1 2 weeks after transduction In non dividing cells e g quiescent CD34 cells or animal tissues e g skeletal muscle neurons transgene expression is more stable and can persist for as long as 6 months following transduction Chen et al 1999 Fan et al 2000 Navarro et al 1999 In actively dividing cells i e doubling time of approximately 24 hours we have found that transgene expression is generally detectable within 24 hours of transduction with maximal expression observed at 48 96 hours 2 4 days post transduction Expression levels generally start to decline by 5 days after transduction In cell lines that exhibit longer doubling times or non dividing cell lines high levels of transgene expression typically persist for a longer time If you are transducing the adenoviral construct into your mammalian cell line for the first time we recommend performing a time course of expression to determine the optimal conditions for expression of your recombinant protein To obtain optimal expression of your gene of interest you will need to transduce the adenoviral construct into your mammalian cell line of choice using a suitable MOI MOT is defined as the number of virus particles per cell and generally correlates with expression Typically expression levels increase linearly as the MOI increases A number of factors can influence determination of an optimal MOI
24. fficient Gene Transfer and Long Term Expression in Neurons Using a Recombinant Adenovirus with a Neuron Specific Promoter Gene Ther 6 1884 1892 Russell W C 2000 Update on Adenovirus and its Vectors J Gen Virol 81 2573 2604 Wang I I and Huang I I 2000 Adenovirus Technology for Gene Manipulation and Functional Studies Drug Discovery Today 5 10 16 Wivel N A 1999 Adenoviral Vectors In The Development of Human Gene Therapy T Friedmann ed Cold Spring Harbor NY Cold Spring Harbor Laboratory Press pp 87 110 Zhang W W Koch P E and Roth J A 1995 Detection of Wild Type Contamination in a Recombinant Adenoviral Preparation by PCR BioTechniques 18 444 447 2002 2005 Invitrogen Corporation All rights reserved 32 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
25. fieation irre Es lates aaaea ees e aae a E ae Ea EE Ea 29 References A TN 31 iii iv Kit Contents and Storage Types of Kits Expression Kit Components Shipping Storage pAd DEST Vectors 293A Cell Line This manual is supplied with the kits listed below Product Catalog no ViraPower Adenoviral Gateway Expression Kit K4930 00 ViraPower Adenoviral Promoterless Gateway Expression Kit K4940 00 The ViraPower Adenoviral Expression Kits include the following components For a detailed description of the contents of each component see below Components Catalog no K4930 00 Catalog no K4940 00 pAd CMV V5 DEST Y Gateway Vector pAd PL DEST Gateway Y Vector 293A Cell Line Y Y The ViraPower Adenoviral Expression Kits are shipped as described below Upon receipt store each component as detailed below Item Shipping Storage pAd DEST Gateway Vector Blue ice 20 C 293A Cell Line Dry ice Liquid nitrogen Each ViraPower Adenoviral Expression Kit includes a destination vector pAd CMV V5 DEST or pAd PL DEST for cloning your DNA sequence of interest and a corresponding expression control vector Refer to the pAd CMV V5 DEST and pAd PL DEST Gateway Vector manual for detailed information about the amount provided and instructions on how to use the vectors The pAd CMV V5 DEST and pAd PL DEST Gateway V
26. hanek S and Clemens P R 1999 DNA from Both High Capacity and First Generation Adenoviral Vectors Remains Intact in Skeletal Muscle Hum Gene Ther 10 365 373 Ciccarone V Chu Y Schifferli K Pichet J P Hawley Nelson P Evans K Roy L and Bennett S 1999 Lipofectamine 2000 Reagent for Rapid Efficient Transfection of Eukaryotic Cells Focus 21 54 55 Dion L D Fang J and R I Garver J 1996 Supernatant Rescue Assay vs Polymerase Chain Reaction for Detection of Wild Type Adenovirus Contaminating Recombinant Adenovirus Stocks J Virol Methods 56 99 107 Engelhardt J F Yang Y Stratford Perricaudet L D Allen E D Kozarsky K Perricaudet M Yankaskas J R and Wilson J M 1993 Direct Gene Transfer of Human CFTR Into Human Bronchial Epithelia of Xenografts with E1 Deleted Adenoviruses Nature Genetics 4 27 34 Fallaux F J Bout A Velde I V d Wollenberg D J V d Hehir K M Keegan J Auger C Cramer S J Ormondt H V Eb A J V d Valerio D and Hoeben R C 1998 New Helper Cells and Matched Early Region 1 Deleted Adenovirus Vectors Prevent Generation of Replication Competent Adenoviruses Hum Gene Ther 9 1909 1917 Fallaux F J Kranenburg O Cramer S J Houweling A Ormondt H V Hoeben R C and Eb A J V d 1996 Characterization of 911 A New Helper Cell Line for the Titration and Propagation of Early Region 1 Deleted
27. he buyer cannot sell or otherwise transfer a this product b its components or c materials made by the employment of this product or its components to a third party or otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes The buyer may transfer information or materials made through the employment of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Notwithstanding the preceding any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a license from Invitrogen under the patents identified above to distribute such materials Transfer of such materials and or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic pur
28. ian cell line of choice 5 Assay for transient expression of your recombinant protein For details and instructions to generate your expression construct using pAd CMV V5 DEST or pAd PL DEST refer to the pAd CMV V5 DEST and pAd PL DEST Gateway Vector manual For instructions to culture and maintain the 293A producer cell line refer to the 293A Cell Line manual These manuals are supplied with the ViraPower Adenoviral Expression Kits and are also available for downloading from our Web site www invitrogen com or by contacting Technical Service see page 26 The ViraPower Adenoviral Expression System Components of The ViraPower Adenoviral Expression System facilitates highly efficient in the ViraPower vitro or in vivo delivery of a target gene to dividing and non dividing mammalian Adenoviral cells using a replication incompetent adenovirus Based on the second Expression generation vectors developed by Bett et al 1994 the ViraPower Adenoviral System Expression System takes advantage of the Gateway Technology to simplify and greatly enhance the efficiency of generating high titer recombinant adenovirus e The first major component of the System is an El and E3 deleted pAd DEST based expression vector into which the gene of interest will be cloned Expression of the gene of interest is controlled by the human cytomegalovirus CMV promoter in pAd CMV V5 DEST or the promoter of choice in pAd PL DEST
29. invitrogen ViraPower Adenoviral Expression System A viral system for high level transient expression in dividing and non dividing mammalian cells Catalog nos K4930 00 and K4940 00 Version B 11 July 2005 25 0543 A Limited Use Label License covers this product see Purchaser N otification By use of this product you accept the terms and conditions of the Limited Use Label License ii Table of Contents Table of Contents cani A Hui iii Kit Contents Storage ra eae ea e E E ER Aa RE eA E OEE v Accessory TPO AU CIS ata ir an arado vi Introduction ci cng acesserece 1 OVERVIEWS un nenne seh nein uni ss ne asp kn 1 The ViraPower Adenoviral Expression System asian een 3 Biosafety Feat r s of the it dia ii 5 Experimental Outline 2 22 ein aa cabs Sen nein ein Idstein tada cn ia 7 o 8 General Informatica RR edi 8 Producing Adenovirus in 293A Cells uennsnssnensnessnsnsnsnsennnnnnnsnnnnnnnnnnnnnnnnennnennnnnnnnnnnnnnnnnnn 9 Amplifying Your Adenoviral Stock siiis i iena E aE aE h R E a a AE EETA RARE RES 14 Titering Your Adenoviral Stockis teeseen eiee Ei E ee Ee are ioa E aie PETE EAEE EE S TEKER EA 17 Transduction and Analysis cinc clado ni aner aA ene EEEa aee REESEN Oae anida ai aaa 21 Troubleshooting sp dae 23 A E BER SARE EL ERE IESIERNUETECERERFDEL EEFFELFPBEPEBELFEEFEEEEHEEFUEBEREFEBEFEBEREUEEFPEPEBRE EEEBERE PUEFEDR 25 NO NO 25 Technical Serie Dee reader 26 Purchaser Notification ii A AAA A toes 27 Product Quali
30. late size 6 well one well per adenoviral construct Number of 293A cells to transfect 5 x 10 cells see Note below Amount of Pac I digested pAd DEST 1 ug expression plasmid Amount of Lipofectamine 2000 3 ul 293A cells should be plated 24 hours prior to transfection in complete medium and Note should be 90 95 confluent on the day of transfection Make sure that cells are healthy at the time of plating continued on next page 10 Producing Adenovirus in 293A Cells continued Transfection Procedure Follow the procedure below to transfect 293A cells using Lipofectamine 2000 Remember that you may keep the cells in culture medium during transfection We recommend including a positive control and a negative control no DNA no Lipofectamine 2000 in your experiment to help you evaluate your results 1 The day before transfection trypsinize and count the 293A cells plating them at 5 x 10 cells per well in a 6 well plate Plate cells in 2 ml of normal growth medium containing serum On the day of transfection remove the culture medium from the 293A cells and replace with 1 5 ml of normal growth medium containing serum or Opti MEM I Medium containing serum Do not include antibiotics Prepare DNA Lipofectamine 2000 complexes for each transfection sample by performing the following e Dilute 1 ug of Pac I digested pAd DEST expression plasmid DNA in 250 ul of Opti MEM I Medium wi
31. line of interest 5 Assay for recombinant protein of interest Methods General Information Important Generating Your pAd DEST Expression Clone 293A Cell Line The ViraPower Adenoviral Expression System is designed to help you create an adenovirus to deliver and transiently express a gene of interest in mammalian cells Although the system has been designed to help you express your recombinant protein of interest in the simplest most direct fashion use of the system is geared towards those users who are familiar with the biology of DNA viruses and adenoviral vectors We highly recommend that users possess a working knowledge of viral and tissue culture techniques For more information about these topics refer to the following published reviews e Adenovirus biology see Russell 2000 e Adenoviral vectors see Hitt et al 1999 and Wivel 1999 e Adenovirus applications see Wang and Huang 2000 You will need to generate an expression clone containing your DNA sequence of interest in pAd CMV V5 DEST or pAd PL DEST If you want to Then use Express your gene of interest under the pAd CMV V5 DEST control of the human CMV promoter Express your gene of interest under the pAd DEST control of your own promoter of choice Refer to the pAd CMV V5 DEST and pAd PL DEST Gateway Vector manual for instructions to create your expression clone Once you have created your expression clone us
32. ls cultured in the appropriate medium see the 293A Cell Line manual for details e Transfection reagent suitable for transfecting 293A cells e g Lipofectamine 2000 see the next page for more information e Opti MEM I Reduced Serum Medium if using Lipofectamine 2000 pre warmed see the next page e Fetal bovine serum FBS e Sterile 6 well and 10 cm tissue culture plates e Sterile tissue culture supplies e 15 mlsterile capped conical tubes e Table top centrifuge e Water bath set to 37 C e Cryovials continued on next page Producing Adenovirus in 293A Cells continued Positive Control The pAd CMV V5 GW lacZ plasmid is included with each ViraPower Adenoviral Expression kit as a positive control vector for expression We recommend including the positive control vector in your transfection experiment to generate a control adenoviral stock that you may use to help you optimize expression conditions in your mammalian cell line of interest To use pAd CMV V5 GW lacZ as a positive control you will need to digest the vector with Pac I using the protocol on the previous page The Pac I digested plasmid may then be used in your transfection experiment to generate an adenoviral stock For more information about the positive control vector refer to the pAd CMV V5 DEST and pAd PL DEST Gateway Vector manual Transfection You may use any suitable transfection reagent to introduce the pAd DEST Reagent exp
33. n Customers 28 The use of the CMV promoter is covered under U S Patent Nos 5 168 062 and 5 385 839 owned and licensed by the University of lowa Research Foundation 214 Technology Innovation Center Iowa City Iowa 52242 Commercial users must obtain a license to these patents directly from the University of lowa Research Foundation For further information please contact the Associate Director of UIRE at 319 335 4546 This product is covered by United States Patent No 6 136 594 under which Invitrogen has been granted a limited right to provide products for research purposes Your use of this product constitutes your agreement to use this product for internal research purposes only and not for any clinical therapeutic prophylactic diagnostic or production use If you do not agree to be bound by these terms return the unopened container s to Invitrogen for a full refund The 293A Cell Line is genetically modified and carries adenovirus type 5 sequences Asa condition of sale this product must be in accordance with all applicable local legislation and guidelines including EC Directive 90 219 EEC on the contained use of genetically modified organisms Product Qualification Introduction Vectors 293A Cell Line Adenovirus Production This section describes the criteria used to qualify the components of the ViraPower Adenoviral Expression Kits Refer to the pAd CMV V5 DEST and pAd PL DEST Gateway V
34. n clone into 293A cells you must expose the left and right viral ITRs to allow proper viral replication and packaging Each pAd DEST vector contains Pac I restriction sites refer to the maps of each vector in the pAd CMV V5 DEST and pAd PL DEST manual for the location of the Pac I sites Digestion of the vector with Pac I allows exposure of the left and right viral ITRs and removal of the bacterial sequences i e pUC origin and ampicillin resistance gene Note Make sure that your DNA sequence of interest does not contain any Pac I restriction sites 1 Digest at least 5 ug of purified plasmid DNA of your pAd DEST expression construct with Pac I New England Biolabs Catalog no R0547S Follow the manufacturer s instructions 2 Purify the digested plasmid DNA using phenol chloroform extraction followed by ethanol precipitation or a DNA purification kit e g Invitrogen s S N A P MiniPrep Kit Catalog no K1900 01 Note Gel purification is not required 3 Resuspend or elute the purified plasmid as appropriate in sterile water or TE Buffer pH 8 0 to a final concentration of 0 1 3 0 ug ul You should have the following materials on hand before beginning e Pac I digested pAd DEST expression clone containing your DNA sequence of interest 0 1 3 0 ug ul in sterile water or TE pH 8 0 e pAd CMV V5 GW lacZ positive control vector supplied with the kit resuspend in sterile water to a concentration of 1 ug yl e 293A cel
35. n com or requested from Technical Service see page 26 Use of the ViraPower Adenoviral Expression System to facilitate DNA virus based expression of the gene of interest provides the following advantages e Uses Gateway Technology to allow highly efficient rapid cloning of a gene of interest into a full length adenoviral vector bypassing the need for a shuttle vector and inefficient homologous recombination in human or bacterial cells e Allows generation of high titer adenoviral stocks i e 1 x 10 pfu ml in crude preparations and 1 x 10 pfu ml in concentrated preparations e Efficiently delivers the gene of interest to actively dividing and non dividing mammalian cells in culture or in vivo e Generates adenoviral constructs with such a high degree of efficiency and accuracy that the system is amenable for use in high throughput applications or library transfer procedures e Allows production of a replication incompetent virus that enhances the biosafety of the system and its use as a gene delivery vehicle Overview continued Purpose of this Manual This manual provides an overview of the ViraPower Adenoviral Expression System and provides instructions and guidelines to 1 Transfect the pAd CMV V5 DEST or pAd PL DEST expression construct into the 293A Cell Line to produce an adenoviral stock Amplify the adenoviral stock Titer the adenoviral stock Use the amplified adenoviral stock to transduce your mammal
36. ng et al 1995 or supernatant rescue assays Dion et al 1996 e If RCA contamination occurs perform plaque purification to re isolate the recombinant adenovirus of interest Note As an alternative El containing producer cell lines such as 911 Fallaux et al 1996 or PER C6 Fallaux et al 1998 which contain no regions of homologous overlap with the adenoviral vectors can be used to help reduce the incidence of RCA generation Experimental Outline Flow Chart The diagram below describes the general steps required to express your gene of interest using the ViraPower Adenoviral Expression System For instructions to generate your adenovirus expression clone using pAd CMV V5 DEST or pAd PL DEST refer to the pad CMV V5 DEST and pAd PL DEST Gateway Vector manual Your Mammalian Cell Line of Interest Promoter gene of interest Adenovirus Expression 293A Producer Cell Line 1 Generate the adenoviral expression clone containing your DNA of interest see the pAd PL DEST and pAd CMV V5 DEST manual for instructions Digest the purified plasmid with Pac I to expose the ITRs 2 Transfect the 293A producer cell line with your adenoviral expression clone Harvest cells and prepare a crude viral lysate 3 Amplify the adenovirus by infecting 293A producer cells with the crude viral lysate Determine the titer of your adenoviral stock 4 Add the viral supernatant to your mammalian cell
37. nstruct we recommend that you titer all of the adenoviral constructs using the same mammalian cell line To determine the titer of your adenoviral construct you should have the following materials on hand before beginning e Your pAd DEST adenoviral stock store at 80 C until use e 293A Cell Line or other appropriate mammalian cell line of choice see above e Complete culture medium for your cell line e 6 well tissue culture plates e 4 agarose see Recipes page 25 equilibrate to 65 C before use e Plaquing media normal growth medium containing 2 FBS equilibrate to 37 C before use e 5 mg ml MTT solution or other appropriate reagent for staining see Recipes page 25 see below for alternatives We recommend using the vital dye 3 4 5 Dimethylthiazol 2 y1 2 5 diphenyltetrazolium bromide Thiazolyl blue MTT as a staining reagent to help visualize plaques Other vital stains including Neutral Red Sigma Catalog no N7005 are suitable If you wish to use Neutral Red prepare a 1 solution 100X stock solution in water and store at 4 C continued on next page Titering Your Adenoviral Stock continued Titering Procedure Follow the procedure below to determine the titer of your adenoviral stock using the 293A Cell Line or other appropriate cell line You will use at least one 6 well plate for every adenoviral stock to be titered six dilutions or one mock well and five dilutions Note If you have generat
38. nucleus Once in the nucleus the early events are initiated e g transcription and translation of El proteins followed by expression of the adenoviral late genes and viral replication Note that expression of the late genes is dependent upon El In the ViraPower Adenoviral Expression System El is supplied by the 293A producer cells The viral life cycle spans approximately 3 days For more information about the adenovirus life cycle and adenovirus biology refer to published reviews Russell 2000 continued on next page The ViraPower Adenoviral Expression System continued Recombinant Protein Expression Infection vs Transduction In vivo Gene Delivery After adenovirus is transduced into the target cell and is transported to the nucleus it does not integrate into the host genome Therefore expression of your recombinant protein of interest e Is typically detectable within 24 hours after transduction e Is transient and will only persist for as long as the viral genome is present For more information see page 21 Note that we refer to viral infection in some procedures in this manual and viral transduction in other procedures These terms are defined below e Infection Applies to situations where viral replication occurs and infectious viral progeny are generated Only cell lines that stably express El can be infected e Transduction Applies to situations where no viral replication occurs and no inf
39. omers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Service Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and ex
40. or efficient packaging see table below Vector Insert Size Limit pAd CMV V5 DEST 6 0 kb pAd PL DEST 7 5 kb e The characteristics of the cell line used for titering see the next page for more information e The age of your adenoviral stock Viral titers may decrease with long term storage at 80 C If your adenoviral stock has been stored for 6 months to 1 year we recommend titering or re titering your adenoviral stock prior to use in an expression experiment e Number of freeze thaw cycles A limited number of freeze thaw cycles is acceptable but viral titers can decrease with more than 10 freeze thaw cycles e Improper storage of your adenoviral stock Adenoviral stocks should be aliquotted and stored at 80 C see page 13 for recommended storage conditions continued on next page 17 Titering Your Adenoviral Stock continued Selecting a Cell Line Note Materials Needed Staining Reagents 18 We recommend using the 293A cell line supplied with the kit to titer your adenoviral stock Other cell lines are suitable If you wish to use another cell line choose one with the following characteristics e Must express the El proteins e Grows as an adherent cell line e Easy to handle e Exhibits a doubling time in the range of 18 25 hours e Non migratory The titer of an adenoviral construct may vary depending on which cell line is chosen If you have more than one adenoviral co
41. poses or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that none of i this product ii any of its components or iii a method claim of the foregoing patents was used in the manufacture of such product Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to use this product for purposes other than those permitted above contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 continued on next page 27 Purchaser Notification continued Limited Use Label License No 28 CMV Promoter Limited Use Label License No 116 Adenoviral Technology Products Information for Europea
42. ression construct into 293A cells We recommend using the cationic lipid based Lipofectamine 2000 Reagent Ciccarone et al 1999 available from Invitrogen see page vi for ordering information Using Lipofectamine 2000 to transfect 293A cells offers the following advantages e Provides the highest transfection efficiency in 293A cells TM e DNA Lipofectamine 2000 complexes can be added directly to cells in culture medium in the presence of serum e Removal of complexes or medium change or addition following transfection are not required although complexes can be removed after 4 6 hours without loss of activity TM Note To facilitate optimal formation of DNA Lipofectamine 2000 complexes we recommend using Opti MEM I Reduced Serum Medium available from Invitrogen For more information about Opti MEM I see our Web site www invitrogen com or call Technical Service see page 26 Recommended We generally produce adenoviral stocks in 293A cells using the following Transfection optimized transfection conditions below The amount of adenovirus produced Conditions using these recommended conditions is approximately 10 ml of crude viral lysate with a titer ranging from 1 x 10 to 1 x 10 plaque forming units pfu ml ging plaq 8 P TM Note We use Lipofectamine 2000 for transfection If you are using another transfection reagent follow the manufacturer s instructions Condition Amount Tissue culture p
43. rom 1 2 to 1 3 If you are using another transfection reagent optimize according to the manufacturer s recommendations Viral supernatant too dilute Concentrate virus using CsCl purification Engelhardt et al 1993 or any method of choice Viral supernatant frozen and thawed multiple times Do not freeze thaw viral supernatant more than 10 times Gene of interest is large Viral titers generally decrease as the size of the insert increases inserts larger than 6 kb for pAd CMV V5 DEST and 7 5 kb for pAd PL DEST are not recommended Gene of interest is toxic to cells Generation of constructs containing activated oncogenes or potentially harmful genes is not recommended No plaques obtained upon titering Viral stocks stored incorrectly Aliquot and store stocks at 80 C Do not freeze thaw more than 10 times Incorrect titering cell line used Use the 293A cell line or any cell line with the characteristics discussed on page 18 Agarose overlay incorrectly prepared Make sure that the agarose is not too hot before addition to the cells hot agarose will kill the cells Titer indeterminable cells completely lysed Viral supernatant not diluted sufficiently Titer adenovirus using 10 fold serial dilutions ranging from 10 to 10 continued on next page 23 Troubleshooting continued Transducing Mammalian Cells 24 The table below lists
44. sistent toxicity in target cells Too much crude viral stock used e Reduce the amount crude viral stock used for transduction or dilute the crude viral stock e Amplify the adenoviral stock e Concentrate the crude viral stock Wild type RCA contamination Screen for RCA contamination Dion et al 1996 Zhang et al 1995 Plaque purify to isolate recombinant adenovirus or prepare a new adenoviral stock Recipes 4 Agarose 5 mg ml MTT Appendix Follow the procedure below to prepare a 4 Agarose solution Materials Needed Ultra Pure Agarose Invitrogen Catalog no 15510 027 Deionized sterile water Protocol 1 Prepare a 4 stock solution in deionized sterile water 2 Autoclave at 121 C for 20 minutes to sterilize 3 Equilibrate to 65 C in a water bath and use immediately or store at room temperature indefinitely If you store the agarose solution at room temperature you will need to melt the agarose before use Microwave the agarose to melt then equilibrate to 65 C in a water bath before use Follow the procedure below to prepare a 5 mg ml MTT solution Materials Needed 3 4 5 Dimethylthiazol 2 y1 2 5 diphenyltetrazolium bromide Thiazolyl blue MTT Sigma Catalog no M2128 Phosphate Buffered Saline PBS Invitrogen Catalog no 10010 023 Protocol 1 Prepare a 5 mg ml stock solution in PBS 2 Filter sterilize and dispense 5 ml aliquots into sterile conical tubes
45. the desired day e g 2 days post transduction and assay for expression of your recombinant protein You may use any method of choice to detect your recombinant protein of interest including functional analysis immunofluorescence or western blot If you have cloned your gene of interest in frame with an epitope tag you may detect your recombinant protein using an antibody to the epitope tag see the pad CMV V5 DEST and pAd PL DEST Gateway Vector manual for details Troubleshooting Introduction Generating the Adenoviral Stock Review the information in this section to troubleshoot your adenoviral expression experiments The table below lists some potential problems and possible solutions that may help you troubleshoot your transfection amplification and titering experiments Problem Reason Solution Low viral titer Low transfection efficiency Incomplete Pac 1 digestion or digested DNA contaminated with phenol ethanol or salts Unhealthy 293A cells cells exhibit low viability 293A cells plated too sparsely on the day before transfection Plasmid DNA transfection reagent ratio incorrect e Repeat the Pac I digestion Make sure purified DNA is not contaminated with phenol ethanol or salts e Use healthy 293A cells do not overgrow e Cells should be 90 95 confluent at the time of transfection e Optimize such that plasmid DNA in ug Lipofectamine 2000 in ul ratio ranges f
46. the mammalian cell line of interest and express your recombinant protein we highly recommend determining the titer of your adenoviral stock While this procedure is not required for some applications it is necessary if e You wish to control the number of adenoviral particles introduced to each cell e You wish to generate reproducible expression results Guidelines and protocols are provided in this section To determine the titer of an adenoviral stock you will 1 Plate 293A cells in 6 well tissue culture plates 2 Prepare 10 fold serial dilutions of your adenoviral stock 3 Infect 293A cells overnight with serial dilutions of adenoviral stock 4 Perform a plaque assay by overlaying the infected 293A cells with an agarose plaquing media solution Allow 8 12 days for plaques to form 5 Stain and count the number of plaques in each dilution A number of factors can influence viral titers including e The size of your gene of interest Titers will generally decrease as the size of the insert increases The size of the wild type adenovirus type 5 genome is approximately 35 9 kb Studies have demonstrated that recombinant adenovirus can efficiently package up to 108 of the wild type virus size from El and E3 deleted vectors Bett et al 1994 Taking into account the size of the elements required for expression from each pAd DEST vector make sure that your DNA sequence or gene of interest does not exceed the size indicated f
47. thout serum Mix gently e Mix Lipofectamine 2000 gently before use then dilute 3 ul in 250 pl of Opti MEM I Medium without serum Mix gently and incubate for 5 minutes at room temperature e After the 5 minute incubation combine the diluted DNA with the diluted Lipofectamine 2000 Mix gently e Incubate for 20 minutes at room temperature to allow the DNA Lipofectamine 2000 complexes to form The solution may appear cloudy but this will not impede the transfection Add the DNA Lipofectamine 2000 complexes dropwise to each well Mix gently by rocking the plate back and forth Incubate the cells overnight at 37 C in a CO incubator The next day remove the medium containing the DNA Lipofectamine 2000 complexes and replace with complete culture medium i e D MEM containing 10 FBS 2 mM L glutamine and 1 penicillin streptomycin 48 hours post transfection trypsinize cells and transfer the contents of each well to a sterile 10 cm tissue culture plate containing 10 ml of complete culture medium Caution Remember that you are working with infectious virus at this stage and in all subsequent procedures Follow the recommended guidelines for working with BL 2 organisms see page 5 for more information Replace culture medium with fresh complete culture medium every 2 3 days until visible regions of cytopathic effect CPE are observed typically 7 10 days post transfection For an example see the next page
48. tock See page 17 and the Troubleshooting section page 23 for more tips and guidelines to optimize your viral yield For some applications viral titers higher than 1 x 10 pfu ml may be desired It is possible to concentrate adenoviral stocks using a variety of methods e g CsCl purification Engelhardt et al 1993 without significantly affecting their transducibility Use of these methods allows generation of adenoviral stocks with titers as high as 1 x 10 pfu ml Transduction and Analysis Introduction Transient Expression Note Multiplicity of Infection MOI Determining the Optimal MOI Once you have generated an adenoviral stock with a suitable titer you are ready to transduce the adenoviral construct into the mammalian cell line of choice and assay for expression of your recombinant protein Guidelines are provided below The pAd CMV V5 DEST or pAd PL DEST adenoviral construct is replication incompetent and does not integrate into the host genome Therefore once transduced into the mammalian cells of choice your recombinant protein of interest will be expressed only as long as the viral genome is present The adenovirus terminal protein TP covalently binds to the ends of the viral DNA and helps to stabilize the viral genome in the nucleus Russell 2000 In actively dividing cells the adenovirus genome is gradually diluted out as cell division occurs resulting in an overall decrease in transgene
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