pBudCE4.1 - Thermo Fisher Scientific
Contents
1. For established cell lines e g HeLa consult original references or the supplier of your cell line for the optimal method of transfection We recommend that you follow exactly the protocol for your cell line Pay particular attention to medium requirements when to pass the cells and at what dilution to split the cells Further information is provided in Current Protocols in Molecular Biology Ausubel et al 1994 Methods for transfection include calcium phosphate Chen and Okayama 1987 Wieler et al 1977 lipid mediated Felgner et al 1989 Felgner and Ringold 1989 and electroporation Chu et al 1987 Shigekawa and Dower 1988 TM Invitrogen offers the Lipofectamine 2000 Reagent for lipid mediated transfection pBudCE4 1 lacZ CAT is provided as a positive control vector for mammalian cell transfection and expression and may be used to optimize transfection conditions for your cell line see page 17 The gene encoding B galactosidase is expressed from the CMV promoter as a fusion to the myc epitope in mammalian cells The gene encoding chloramphenicol acetyltransferase CAT is expressed as a fusion to the V5 epitope from the EF 1a promoter A successful transfection results in B galactosidase and CAT expression that can be easily assayed see page 9 Continued on next page Transfection and Analysis Continued Detecting Fusion Proteins Polyacrylamide Gel Electrophoresis Antibodies are available2. l l 3109 GGC TGG GCC CGT TTC GAA GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT Gly Trp Ala Arg Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Polyhistidine 6xHis tag l l l 3157 CTC GAT TCT ACG CGT ACC GGT CAT CAT CAC CAT CAC CAT TGA G Leu Asp Ser Thr Arg Thr Gly His His His His His His BGH Reverse priming site l 3200 TTTAAACCCG CTGATCAGCC TCGACTGTGC CTTCTAGTTG CCAGCCATCT GTTGTTTGCC BGH polyadenylation signal rd 3260 CCTCCCCCGT GCCTICCTTG ACCCTGGAAG GTGCCACTCC CACTGTCCTT TCCTAATAAA 3320 ATGAGGAAAT TGCATCGCAT TGTCTGAGTA GGTGTCATTC TATTCTGGGG GGTGGGGTGG 3380 GGCAGGACAG CAAGGGGGAG GATTGGGAAG ACAATAGCAG GCATGCTGGG GATGCGGTGG Note that there are two BstB I sites in the polylinker Continued on next page Cloning into pBudCE4 1 Continued E coli Transformation EN Y Ad ECO Nor Preparing a Glycerol Stock Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10 and select on Low Salt LB plates containing 25 50 pg mL Zeocin see page 20 Select 10 20 clones and analyze for the presence and orientation of your insert We recommend that you sequence your construct to confirm that each of your genes is fused in frame with the C terminal peptide Several primers are available separately that you may use to sequence your construct These are marked in the multiple cloning site diagrams on pages 4 5 For ordering information see pag
3. pUC origin High copy number replication and growth in E coli pBudCE4 1 lacZ CAT Description pBudCE4 1 lacZ CAT is an 8432 bp control vector containing the gene for B galactosidase and the gene for chloramphenicol acetyltransferase CAT The lacZ gene was excised from pIND lacZ using Hind III and Xba I and cloned into Hind I Xba I digested pBudCE4 1 The CAT gene was cloned by digesting pBudCE4 1 lacZ and pBudCE4 lacZ CAT with Bgl ll and Mun I A fragment containing the CAT gene and part of the EF 1a promoter from pBudCE4 lacZ CAT was cloned into Bgl U Mun I digested pBudCE4 1 lacZ to generate pBudCE4 1 lacZ CAT Map of Control The figure below summarizes the features of the pBudCE4 1 lacZ CAT vector Vector The nucleotide sequence for pBudCE4 1 lacZ CAT is available for downloading from our website www invitrogen com or by contacting Technical Support see page 23 Hind III pBudCE4 1 lacZ CAT Comments for pBudCE4 1 lacZ CAT 8432 nucleotides CMV promoter bases 7 594 CMV Forward priming site bases 544 564 T7 promoter priming site bases 638 657 LacZ fusion bases 673 3966 LacZ ORF bases 673 3840 myc epitope bases 3901 3963 6xHis tag bases 3946 3963 SV40 polyadenylation sequence bases 3986 4116 Zeocin resistance gene bases 4245 4619 complementary strand EM7 promoter bases 4638 4693 complementary strand SV40 early promoter bases 4728 5051 complementary strand EF la pro
4. Mizushima S and Nagata S 1990 pEF BOS a Powerful Mammalian Expression Vector Nucleic Acids Res 18 5322 Mulsant P Tiraby G Kallerhoff J and Perret J 1988 Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells Somat Cell Mol Genet 14 243 252 Nelson J A Reynolds Kohler C and Smith B A 1987 Negative and Positive Regulation by a Short Segment in the 5 Flanking Region of the Human Cytomegalovirus Major Immediate Early Gene Molec Cell Biol 7 4125 4129 Neumann J R Morency C A and Russian K O 1987 A Novel Rapid Assay for Chloramphenicol Acetyltransferase Gene Expression BioTechniques 5 444 447 Perez P Tiraby G Kallerhoff J and Perret J 1989 Phleomycin Resistance as a Dominant Selectable Marker for Plant Cell Transformation Plant Mol Biol 13 365 373 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells BioTechniques 6 742 751 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 15
5. 80 90 confluent 3 Harvest the cells by treating with trypsin EDTA for 2 to 5 minutes or by scraping the cells in PBS 4 Inactivate the trypsin by diluting with fresh medium if necessary and transfer the cells to a sterile microcentrifuge tube 5 Centrifuge the cells at 1 500 rpm for 5 minutes Resuspend the cell pellet in PBS 6 Centrifuge the cells at 1 500 rpm for 5 minutes You may lyse the cells immediately or freeze in liquid nitrogen and store at 80 C until needed TM TM If you are using ProBond resin refer to the ProBond Purification System manual for details about sample preparation for chromatography The ProBond Purification System manual is available for downloading at our website www invitrogen com or by contacting Technical Support see page 23 TM If you are using other metal chelating resin refer to the manufacturer s instruction for recommendations on sample preparation Appendix Human EF 1a Promoter Description The diagram below shows the features of the EF 1a promoter used in pBudCE4 1 Mizushima and Nagata 1990 Features are marked as per Uetsuki et al 1989 Uetsuki et al 1989 7 5 end of human EF la promoter AGCTAGCTTC GTGAGGCTCC GGTGCCCGTC AGTGGGCAGA GCGCACATCG CCCACAGTCC CCGAGAAGTT GGGGGGAGGG TAAACTGGGA AAGTGATGTC TATA box Lal CGTATATAAG TGCAGTAGTC A 5 end of Intron 1 CACAGGTAAG GCGTGCCTTG TTGGAAGTGG GAGTTGAGGC CCTGTCTCGC CGCT
6. HO OH OH Zeocin is used for selection in mammalian cells Mulsant et al 1988 plants Perez et al 1989 yeast Baron et al 1992 and prokaryotes Drocourt et al 1990 Suggested concentrations of Zeocin for selection in mammalian cell lines and E coli are listed below Organism Zeocin Concentration and Selective Medium E coli 2550 ng mL in Low Salt LB medium see page 20 for recipe Mammalian Cells 50 1 000 pg mL varies with cell line Efficient selection requires that the concentration of NaCl be no more than 5 g liter lt 90 mM Continued on next page Zeocin Continued Handling Zeocin TM High salt and acidity or basicity inactivates Zeocin Therefore we recommend that you reduce the salt in bacterial medium and adjust the pH to 7 5 to keep the drug active see next page Note The salt concentration should not be adjusted for mammalian cells Changes to the salt concentration are detrimental to cells TM Store Zeocin at 20 C and thaw on ice before use Zeocin is light sensitive Store drug plates and medium containing drug in the dark Wear gloves a laboratory coat and safety glasses or goggles when handling solutions containing Zeocin Zeocin is toxic Do not ingest or inhale solutions containing the drug 19 Recipes Low Salt LB Medium with Zeocin Cell Lysis Buffer 20 For Zeocin to be active the
7. Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 Most E coli strains are suitable for the growth of this vector including TOP10 and DH5a T18 We recommend that you propagate vectors containing inserts in E coli strains that are recombination deficient recA and endonuclease A deficient endA See below for an important note about E coli strains For your convenience TOP10 and DH5a T1 are available from Invitrogen as chemically competent or electrocompetent cells TOP10 only in One Shot format see page 21 Any E coli strain that contains the complete Tn5 transposable element Le DH5 FO SURE SURE2 encodes the ble bleomycin resistance gene These strains will confer resistance to Zeocin We recommend that you choose an E coli strain that does not contain the Tn5 gene i e TOP10 You may use your method of choice for transformation Chemical transformation is the most convenient for most researchers Electroporation is the most efficient and the method of choice for large plasmids To propagate and maintain the pBudCE4 1 vector use a small amount of the supplied 0 5 ug L stock solution in TE pH 8 0 to transform a recA endA E coli strain like TOP10 or equivalent Select transformants on Low Salt LB plates containing 25 50 pg mL Zeocin see page 20 Be sure to prepare a glycerol stock of each plasmid for long term storage see page 6 Your insert shoul
8. TGGTTCATTC TGA 5 end of Exon 2 13 pBudCE4 1 Vector Map of pBudCE4 1 The figure below summarizes the features of the pBudCE4 1 vector The vector sequence is available for downloading from our website www invitrogen com or by contacting Technical Support see page 23 Hind III Bal Il Xho Pst Sse8387 BstX Sal Kpn Acc BstB Sca Comments for pBudCE4 1 4595 nucleotides CMV promoter bases 7 594 Nhe CMV Forward priming site bases 544 564 T7 promoter priming site bases 638 657 CMV multiple cloning site bases 664 713 myc epitope bases 719 748 6xHis tag bases 764 782 SV40 polyadenylation sequence bases 803 933 Zeocin resistance gene bases 1063 1437 complementary strand EM7 promoter bases 1456 1510 complementary strand SV40 early promoter bases 1547 1869 complementary strand EF la promoter bases 1885 3051 EF la Forward priming site bases 2999 3019 EF la multiple cloning site bases 3062 3126 V5 epitope bases 3127 3168 6xHis tag bases 3178 3195 BGH Reverse priming site bases 3218 3235 complementary strand BGH polyadenylation sequence bases 3224 3447 pUC origin bases 3521 4194 Continued on next page 14 pBudCE4 1 Vector Continued pBudCE4 1 4595 bp contains the following elements All features have been functionally tested Feature Benefit Human cytomegalovirus CMV immediate early promoter enhancer Permits effic
9. mL 2 Bring the volume up to 90 mL with deionized water and adjust the pH to 7 8 with HCI 3 Bring the volume up to 100 mL Store at room temperature Note Protease inhibitors may be added fresh at the following concentrations 1 mM PMSF 1 pg mL pepstatin 1 ug mL leupeptin Accessory Products Introduction The products listed below are designed for use with pBudCE4 1 For details visit www invitrogen com or contact Technical Support page 23 Item Quantity Catalog no One Shot TOP10 Chemically Competent E coli 21 x 50 uL C4040 03 One Shot TOP10 Electrocomp Cells 21 x 50 uL C4040 52 One Shot Max Efficiency DH5a TIR 20 x 50 pL 12297 016 PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 Lipofectamine 2000 Reagent 1 5 mL 11668 019 B Gal Assay Kit 80 mL K1455 01 B Gal Staining Kit 1 kit K1465 01 ProBond Purification System 6 purifications K850 01 ProBond Resin ae sete 150 mL R801 15 pene 1 gram R250 01 5 grams R250 05 imMedia Zeo Liquid 200 mL Q620 20 imMedia Zeo Agar 8 10 agar plates Q621 20 WesternBreeze Chromogenic Kit Anti Mouse 1 kit WB7103 WesternBreeze Chromogenic Kit Anti Rabbit 1 kit WB7105 WesternBreeze Chromogenic Kit Anti Goat 1 kit WB7107 WesternBreeze Chemiluminescent Kit Anti 1 kit WB7104 Mouse WesternBreeze Chromogenic Kit Anti Ra
10. salt concentration of the medium must remain low lt 90 mM and the pH must be 7 5 For selection in E coli it is imperative that you prepare LB broth and plates using the following recipe Note the lower salt content of this medium Failure to use low salt LB medium will result in non selection due to inactivation of the drug For more information about Zeocin refer to page 18 Low Salt LB Medium 10 g Tryptone 5 g NaCl 5 g Yeast Extract 1 Combine the dry reagents above and add deionized distilled water to 950 mL Adjust pH to 7 5 with 5 M NaOH Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving Autoclave on liquid cycle at 15 lbs sq in and 121 C for 20 minutes Thaw Zeocin on ice and vortex before removing an aliquot Allow the medium to cool to at least 55 C before adding the Zeocin to 25 50 pg mL final concentration 5 Store plates at 4 C in the dark Plates containing Zeocin are stable for 1 2 weeks For your convenience Low Salt LB medium containing 25 ug ml Zeocin is available as premixed pre sterilized E coli growth medium imMedia that contains everything you need in a convenient pouch Liquid and agar media are available depending upon your application see page 21 50 mM Tris pH 7 8 150 mM NaCl 1 Nonidet P 40 1 This solution can be prepared from the following common stock solutions For 100 ml combine 1 M Tris base 5mL 5 M NaCl 3 mL Nonidet P 40 1
11. 57 Uetsuki T Naito A Nagata S and Kaziro Y 1989 Isolation and Characterization of the Human Chromosomal Gene for Polypeptide Chain Elongation Factor 1a J Biol Chem 264 5791 5798 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 26 invitrogen by Lefe technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
12. Invitrogen by technologies pBudCE4 1 Catalog no V532 20 Rev Date 7 July 2010 Manual part no 25 0389 MANO0000206 ii Table of Contents Kit Contents and Storage svn senses Pe telenet edere enden iv Introduction 2 cssccsscssectsscssecssscssectnecesncsnedesscizecednccescesecceececescestecesineceseccneceiestuesecescoestoeeces 1 ee RRE 1 Experimental TE 2 Methods vou annen eneen enden eed 3 Clonings into PBUdCEA dieran ias n 3 Transfection and Analysis EEN 7 Creating Stable Cell Limes EEN 10 ADP SME O 13 Humar EF Ta Promote ciar rra 13 PBudEE41 EE 14 pBudCR4 Lal CA EE 17 LEDE nnee Ee 18 Kee A TNA AT BG es ea BPs Goto ae 20 e IC 21 Technical Support antuan EA eebe eh o ee 23 Purch ser Notifica usteet 24 References union atada 25 iii Kit Contents and Storage Shipping and Storage Kit Contents pBudCE4 1 vectors are shipped on wet ice Upon receipt store vectors at 20 C All vectors are supplied as detailed below Store the vectors at 20 C Vector Composition Amount pBudCE4 1 40 uL of 0 5 ug L vector in 10 mM Tris 20 ug HCI 1 mM EDTA pH 8 0 pBudCE4 1 lacZ CAT 40 uL of 0 5 ug uL vector in 10 mM Tris 20 ug HCL 1 mM EDTA pH 8 0 Introduction Product Overview pBudCE4 1 Note pBudCE4 1 is a 4 6 kb vector designed for simultaneous expression of two genes in mammalian cell lines The vector contains the human cytomegalovirus CMV immediate early
13. TCC Pro Ser Leu His Ser Sal I Pst l Sse8387 Ace I Seal Xhal Baril fo TGC AGG TCG ACA TCG ATC TTA AGC AGT ACT TCT AGA GGA TCC GAA CAA AAA Cys Arg Ser Thr Ser Ile Leu Ser Ser Thr Ser Arg Gly Ser Glu Gln Lys myc epitope Polyhistidine 6xHis tag CTC ATC TCA GAA GAG GAT CTG AAT ATG CAT ACC GGT CAT CAT CAC CAT CAC Leu Ile Ser Glu Glu Asp Leu Asn Met His Thr Gly His His His His His CAT TGA GTTTGA TCCCCGGGAA TTCAGACATG ATAAGATACA TTGATGAGTT TGGACAAACC His 44 ACAACTAGAA TGCAGTGAAA AAAATGCTTT ATTTGTGAAA TTTGTGATGC TATTGCTTTA SV40 polyadenylation signal A TTTGTAACCA TTATAAGCTG CAATAAACAA GTTGGGGTGG GCGAAGAACT Continued on next page Cloning into pBudCE4 1 Continued EF 1a Multiple Below is the multiple cloning site of pBUDCE4 1 located downstream of the EF 1a Cloning Site promoter Restriction sites are labeled to indicate the cleavage site The promoter is marked using the convention of Uetsuki et al 1989 For more information see page 13 Sequencing primers are available separately see page 22 E 2940 GCACTTGATG TAATTCTCGT TGGAATTTGC CCTTTTTGAG TTTGGATCTT GGTTCATTCT EF lo Forward priming site E Intron 1 l 3000 CAAGCCTCAG ACAGTGGTTC AAAGTTTTTT TCTTCCATTT CAGGTGTCGT GAACACGTGG 5 end of hEF 1a Exon 2 Not I BstB P Kpn 1 BstX I Xho Bel II 3060 T CGC GGC CGC TTC GAA GGT ACC AGC ACA GTG GAC TCG AGA GAT CTG GCC Arg Gly Arg Phe Glu Gly Thr Ser Thr Val Asp Ser Arg Asp Leu Ala V5 epitope Sfi I Bs B I
14. TITITI CGGTTTTTGG Sp 1 TGCCGTGTGT AATTACTTCC GTGGGAGAGT CTGGCCTGGG TGCTTTCGAT CTGGCAAGAT GTCGGCAATT GTGTACTGGC GCCGTGAACG GGTTCCCECG ACCTGGCTGC TCGAGGCCTT CGCTGGGGCC AAGTCTCTAG AGTCTTGTAA Sp 1 GGCCGCGGGC GGCGACGGGG GAACCGGTGC CTAGAGAAGG TCCGCCTTTT TCCCGAGGGT Start of Transcription TICTTTTTCG CAACGGGTTT TGGCGCGGGG GGGGGAGAAC GCCGCCAGAA GCGAGCGCGG CCACCGAGAA GGCCTGGCCT AGTACGTGAT GCGCTTAAGG GCCGCGTGCG CCATTTAAAA ATGCGGGCCA CCCGTGCGTC TCGGACGGGG CTTTACGGGT TCTITGATCCE AGCCCCTTCG AATCTGGTGG TTTTTGATGA AGATCTGCAC CCAGCGCACA GTAGTCTCAA Exon I TATGGCCCTT GAGCTTCGGG CCTCGTGCTT CACCTTICGCG CCTGCTGCGA ACTGGTATTT TGTTCGGCGA GCT TGGCCGGC eeceeeckeer Sp 1 Sp 1 CTGCTCTGGT CCCGGTCGGC GCTCAAAATG AAAGGGCCTT CCAGGCACCT GGTTTTATGC GGCACTTGAT TCAAGCCTCA GCCTGGCCTC ACCAGTTGCG GAGGACGCGG TCCETCCTCA CGATTAGTTC GATGGAGTTT GTAATTCTCC GACAGTGGTT GCGCCGCCGT TGAGCGGAAA CGCTCGGGAG GCCGTCGCTT TCGAGCTTTT CCCCACACTG TTGGAATTTG CAAAGTTTTT GTATEGCCCC GECCFGGGCE GCAAGGCTGG GATGGCCGCT TCCCGGCCCT Sp 1 AGCGGGCGGG TGAGTCACCC Ap 1 CATGEGACTC CACGGAGTAC GGAGTACGTC GTCTTTAGGT AGTGGGTGGA GACTGAAGTT CCCTTTTTGA GTTTGGATCT 3 end of Intron 1 TTCTTCCATT TCAGGTGTCG GCTGCAGGGA ACACAAAGGA CGGGCGCCGT TGGGGGGAGG AGGCCAGCTT
15. bbit 1 kit WB7106 WesternBreeze Chromogenic Kit Anti Goat 1 kit WB7108 Fast Cat Chloramphenicol Acetyltransferase 1 kit F2900 Assay Kit Continued on next page 21 Accessory Products Continued Primers For your convenience Invitrogen offers a custom primer synthesis service Visit www invitrogen com for more details Antibodies and If you do not have an antibody specific to your protein Invitrogen offers the Western Detection Anti myc Anti V5 or Anti His C term antibodies to detect your recombinant Kits fusion protein Horseradish peroxidase HRP and alkaline phosphatase AP conjugated antibodies are available for convenient one step detection Antibody Epitope Catalog no Anti myc Detects a 10 amino acid epitope R950 25 Anti myc HRP derived from c myc Evan et al 1985 R951 25 KLISEEDL Anti myc AP EAD R952 25 Anti V5 Detects a 14 amino acid epitope R960 25 Anti V5 HRP derived from the P and V proteins of R961 25 the paramyxovirus SV5 Southern et al 1991 R962 25 Anti V5 AP E E GKPIPNPLLGLDST Anti His C term Detects the C terminal polyhistidine R930 25 Anti His C term HRP tag requires the free carboxyl group R931 25 NS for detection Lindner et al 1997 EEE Anti His C term AP HHHHHH COOH 932 25 22 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical reso
16. bp Location Supplier Nhe I 1877 Upstream of EF 1a promoter Many BspH I 4240 Backbone New England Biolabs Fsp I 4547 Backbone Many Pvu l 4568 Backbone Many Selection of Stable Integrants Once the appropriate Zeocin concentration is determined you can generate a stable cell line with your construct 1 Transfect your cells using the appropriate protocol for your cell line Include a sample of untransfected cells as a negative control After transfection wash the cells once with 1X PBS and add fresh medium to the cells 48 hours after transfection split the cells into fresh medium no Zeocin and allow cells to attach TM Remove medium and add medium containing Zeocin at the appropriate concentration for your cell line Split the cells such that they are no more than 25 confluent Replenish selective medium every 3 4 days until Zeocin resistant colonies are detected Pick and expand colonies Continued on next page 11 Creating Stable Cell Lines Continued Preparing Cells for Lysis Lysis of Cells 12 Use the procedure below to prepare cells for lysis prior to purification of your protein on ProBond You will need 5 x 10 to 1 x 107 cells for purification of your TM TM protein on a 2 mL ProBond column see ProBond Purification System manual 1 Seed cells in either five T 75 flasks or 2 to 3 T 175 flasks 2 Grow the cells in selective medium until they are
17. d contain a Kozak consensus sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 Kozak 1990 An example of a Kozak consensus sequence is provided below Other sequences are possible but the G or A at position 3 and the G at position 4 shown in bold illustrates the most commonly occurring sequence with strong consensus Replacing one of the two bases at these positions provides moderate consensus while having neither results in weak consensus The ATG initiation codon is shown underlined G A NNATGG If you wish to express your protein WITHOUT the C terminal peptide be sure to include a stop codon Continued on next page Cloning into pBudCE4 1 Continued CMV Multiple Below is the multiple cloning site of pBUDCE4 1 located downstream of the CMV Cloning Site promoter Restriction sites are labeled to indicate the cleavage site Potential stop 501 561 621 677 728 779 841 901 codons are underlined The arrow indicates the predicted start of transcription using T7 RNA polymerase Sequencing primers are available separately see page 22 CMV Forward priming site CAACGGGACT TTCCAAAATG TCGTAACAAC TCCGCCCCAT TGACGCAAAT GGGCGGTAGG CAAT TATA 3 end of CMV Putative start of transcription CGTGTACGGT GGGAGGTCTA TATAAGCAGA GCTCTCTGGC TAACTAGAGA ACCCACTGCT T7 promoter priming site y Hind HI l TACTGGCTTA TCGAAATTAA TACGACTCAC TATAGGGAGA C CCA AGC TTG CAT
18. e 22 Alternatively you may design your own primers for sequencing Once you have identified the correct clone be sure to purify the colony and make a glycerol stock for long term storage It is also a good idea to keep a DNA stock of your plasmid at 20 C 1 Streak the original colony out on a Low Salt LB plate containing 25 pg mL Zeocin Incubate the plate at 37 C overnight Isolate a single colony and inoculate into 1 2 mL of Low Salt LB containing 25 ug mL Zeocin Grow the culture to stationary phase ODsoo 1 2 Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial 6 Store at 80 C Transfection and Analysis Introduction Plasmid Preparation Methods of Transfection Positive Control Once you have confirmed that your inserts are in the correct orientation and fused in frame with the C terminal peptide if desired you are ready to transfect your cell line of choice We recommend that you include the positive control vector and a mock transfection to evaluate your results Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride Contaminants will kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HiPure Miniprep Kit or TM the PureLink HiPure Midiprep Kit see page 21 for ordering information
19. e cell lines using Zeocin We recommend that you test the sensitivity of your mammalian host cell to Zeocin as natural resistance varies among cell lines General information and guidelines are provided below for your convenience For more TM information about Zeocin refer to page 18 The method of killing with Zeocin is quite different from neomycin G418 and hygromycin Cells do not round up and detach from the plate Sensitive cells will TM exhibit the following morphological changes upon exposure to Zeocin e Vast increase in size similar to the effects of cytomegalovirus infecting permissive cells e Abnormal cell shape e Presence of large empty vesicles in the cytoplasm breakdown of the endoplasmic reticulum and Golgi apparatus or scaffolding proteins e Breakdown of plasma and nuclear membrane appearance of many holes in these membranes e Eventually these cells will completely break down and only strings of protein will remain e Zeocin resistant cells should continue to divide at regular intervals to form distinct colonies There should not be any distinct morphological changes in Zeocin resistant cells when compared to cells not under selection with Zeocin To generate a stable cell line expressing your protein you need to determine the minimum concentration of Zeocin required for killing your untransfected host cell line Typically concentrations between 50 and 1 000
20. for 5 minutes 7 Load 20 ug of lysate onto an SDS PAGE gel see below and electrophorese Use the appropriate percentage of acrylamide to resolve your fusion protein To facilitate separation of your recombinant protein by polyacrylamide gel electrophoresis a wide range of pre cast Novex NuPAGE and Tris Glycine polyacrylamide gels and electrophoresis apparatus are available from Invitrogen The patented Novex NuPAGE Gel System prevents the protein modifications associated with Laemmli type SDS PAGE ensuring optimal separation for protein analysis In addition Invitrogen also carries a large selection of molecular weight protein standards and staining kits for visualization of recombinant proteins For more information about the appropriate gels standards and stains to use to visualize your recombinant protein refer to our website www invitrogen com or call Technical Support see page 23 Continued on next page Transfection and Analysis Continued Western Analysis Note Assay for B galactosidase Activity Assay for CAT Activity Purifying Cells To detect expression of your recombinant fusion protein by western blot analysis you may use the Anti myc Anti V5 or the Anti His C term antibodies available from Invitrogen see page 22 for ordering information or an antibody to your protein of interest The ready to use WesternBreeze Chromogenic Kits and WesternBreeze Chemiluminescent Kits are available
21. from Invitrogen to detect expression of fusion proteins from pBudCE4 1 see page 22 In pBudCE4 1 lacZ CAT B galactosidase and CAT are expressed as fusion proteins to the myc epitope or the V5 epitope respectively In addition you may assay for activity of either control protein using one of the assays described on the next page To detect fusion protein by western blot you will need to prepare a cell lysate from transfected cells We recommend that you perform a time course to optimize expression of the fusion protein e g 24 48 72 hours etc after transfection To lyse cells 1 Wash cell monolayers 10 cells once with phosphate buffered saline PBS 2 Scrape cells into 1 mL PBS and pellet the cells at 1 500 x g for 5 minutes 3 Resuspend in 50 pL Cell Lysis Buffer see recipe on page 20 Other lysis buffers may be suitable 4 Incubate cell suspension at 37 C for 10 minutes to completely lyse the cells Note You may prefer to lyse the cells at room temperature or on ice if degradation of your proteins are a potential problem 5 Centrifuge the cell lysate at 10 000 x g for 10 minutes at room temperature to pellet nuclei and transfer the supernatant to a fresh tube Assay the lysate for protein concentration Note Do not use protein assays utilizing Coomassie Blue or other dyes NP 40 interferes with the binding of the dye with the protein 6 Add SDS PAGE sample buffer to a final concentration of 1X and boil the sample
22. from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods see page 21 for ordering For more information refer to our website www invitrogen com or call Technical Support see page 23 The C terminal peptide containing the myc epitope and the polyhistidine tag or the V5 epitope and polyhistidine tag will add approximately 3 kDa to the size of your protein You may assay for B galactosidase expression by activity assay using cell free lysates Miller 1972 or by staining the cells for activity Invitrogen offers the B Gal Assay Kit and the B Gal Staining Kit for fast and easy detection of B galactosidase expression see page 21 You may assay for CAT expression by ELISA assay western blot analysis fluorometric assay or radioactive assay Ausubel et al 1994 Neumann et al 1987 The CAT assay kit is available from Invitrogen for detection of CAT protein see page 21 You will need 5 x 106 to 1 x 107 transfected cells for purification of your protein on a 2 mL ProBond column or other metal chelating column Refer to the manufacturer s instructions before attempting to purify your fusion protein To prepare cells for lysis refer to the protocol on page 20 Creating Stable Cell Lines Introduction Effect of Zeocin on Sensitive and Resistant Cells Selection in Mammalian Cell Lines 10 pBudCE4 1 contains the Zeocin resistance gene for selection of stabl
23. ient high level expression of recombinant protein Andersson et al 1989 Boshart et al 1985 Nelson et al 1987 CMV Forward priming site Permits sequencing through the insert from the 5 end T7 promoter priming site Permits sequencing through the insert from the 5 end Allows for in vitro transcription in the sense orientation CMV Multiple cloning site Seven unique sites allow insertion of your gene myc epitope Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Allows detection of your recombinant protein with the Anti myc Antibody Anti myc HRP Antibody or Anti myc AP Antibody Evan et al 1985 see page 22 for ordering C terminal polyhistidine 6xHis tag Permits purification of your recombinant protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody the Anti His C term HRP Antibody or the Anti His C term AP Antibody Lindner et al 1997 see page 22 for ordering SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA Note The SV40 late polyadenylation signal terminates transcription for the gene cloned into the CMV MCS while the SV40 early polyadenylation signal terminates transcription for the Zeocin resistance gene The signals are encoded on opposite strands in the same fragment of DNA Zeocin resistance gene Selection of tran
24. moter bases 5067 6236 EF la Forward priming site bases 6181 6201 CAT fusion bases 6268 7032 CAT ORF bases 6268 6924 V5 epitope bases 6964 7005 6xHis tag bases 7015 7032 BGH Reverse priming site bases 7055 7072 complementary strand BGH polyadenylation sequence bases 7061 7285 pUC origin bases 7358 8031 17 Zeocin Zeocin Molecular Weight Formula and Structure Applications of Zeocin 18 Zeocin is a member of the bleomycin phleomycin family of antibiotics isolated from Streptomyces Antibiotics in this family are broad spectrum antibiotics that act as strong anti bacterial and anti tumor drugs They show strong toxicity against bacteria fungi including yeast plants and mammalian cells The Zeocin resistance protein has been isolated and characterized Calmels et al 1991 Drocourt et al 1990 This protein the product of the Sh ble gene Streptoalloteichus hindustanus bleomycin gene is a 13 7 kDa protein that binds Zeocin in a stoichiometric manner to inhibit its DNA strand cleavage activity Expression of this protein in eukaryotic and prokaryotic hosts confers resistance TM to Zeocin The formula for Zeocin is CssHs6O21N20S2Cu HCl and the molecular weight is 1 527 5 daltons Zeocin is an HCI salt The diagram below shows the structure of TM o CH3 N R Ho N s G NH o LA N CH S H H H Zeocin CH HO l Faso AAA N NH o R HN aw NH OH O
25. nty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 23 Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 60 EF 1a Promoter 24 This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Switzerland and is provided only for use in research Information about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany EF 1alpha promoter products are sold under license for research purposes only The use of this product for any commercial purpose including but not limited to use in any study for the purpose of a filing of a new drug application requires a license from Mochida Pharmaceutical Co Ltd 7 Yotsuya 1 Chome Shinjuku Ku Tokyo 160 Japan Tel 81 3 3225 5451 Fax 81 3 3225 6091 References Andersson S Davis D L Dahlback H J rnvall H and Russell D W 1989 Cloning Structure and Expression of the Mitochondrial Cytochrome P 450 Sterol 26 Hydroxylase a Bile Acid Biosynthetic Enzyme J Biol Chem 264 8222 8229 Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocol
26. promoter and the human elongation factor 1a subunit EF 1a promoter for high level constitutive independent expression of two recombinant proteins see page 13 for more information on the EF 1a promoter Features of the vector allow detection and purification of expressed proteins see pages 15 16 for more information High level stable and transient expression studies can be carried out in most mammalian cell types In addition to the two promoters the vector contains the following elements e C terminal peptides encoding the myc c myc epitope or the V5 epitope and a polyhistidine 6xHis metal binding tag for detection and purification of recombinant proteins e Zeocin resistance gene for selection in E coli and creation of stable mammalian cell lines Mulsant et al 1988 see pages 18 19 for more information e _SV40 origin for episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen e g COS7 pBudCE4 1 lacZ CAT is included for use as a positive control for transfection expression and detection in the cell line of choice pBudCE4 1 is an improved version of pBudCE4 During construction of the original vector an ATG was inadvertently created in the multiple cloning site 672 674 bp 3 to the CMV promoter Since it may interfere with proper translation of the cloned gene this ATG was changed to ATT to create pBudCE4 1 Continued on next page Experimental Ou
27. s in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Baron M Reynes J P Stassi D and Tiraby G 1992 A Selectable Bifunctional B Galactosidase Phleomycin resistance Fusion Protein as a Potential Marker for Eukaryotic Cells Gene 114 239 243 Boshart M Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus Cell 41 521 530 Calmels T Parriche M Burand H and Tiraby G 1991 High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance Curr Genet 20 309 314 Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Molec Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nucleic Acids Res 15 1311 1326 Drocourt D Calmels T P G Reynes J P Baron M and Tiraby G 1990 Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance Nucleic Acids Res 18 4009 Evan G I Lewis G K Ramsay G and Bishop V M 1985 Isolation of Monoclonal Antibodies Specific for c myc Proto oncogene Product Mol Cell Biol 5 3610 3616 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Tran
28. sfection Proc West Pharmacol Soc 32 115 121 Felgner P L a and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Goldman L A Cutrone E C Kotenko S V Krause C D and Langer J A 1996 Modifications of Vectors pEF BOS pcDNA1 and pcDNA3 Result in Improved Convenience and Expression BioTechniques 21 1013 1015 Goodwin E C and Rottman F M 1992 The 3 Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements Required for Efficient and Accurate Polyadenylation J Biol Chem 267 16330 16334 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biology 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 Miller J H 1972 Experiments in Molecular Genetics Cold Spring Harbor New York Cold Spring Harbor Laboratory Continued on next page 25 References Continued
29. sformants in E coli and stable transfectants in mammalian cells Drocourt et al 1990 Mulsant et al 1988 EM7 promoter Synthetic promoter based on the bacteriophage T7 promoter for expression of the Zeocin resistance gene in E coli Continued on next page 15 pBudCE4 1 Vector Continued Features of pBudCE4 1 Continued 16 Feature Benefit SV40 early promoter and origin Allows efficient high level expression of the Zeocin resistance gene and episomal replication in cells expressing the SV40 large T antigen Human elongation factor 1a EF 10 promoter Permits efficient high level expression of recombinant protein Goldman et al 1996 Mizushima and Nagata 1990 EF 1a Forward priming site Permits sequencing through the insert from the 5 end EF 1a Multiple cloning site Seven unique sites allow insertion of your gene V5 epitope Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Allows detection of your recombinant protein with the Anti V5 Antibody Anti V5 HRP Antibody or the Anti V5 AP Antibody Southern et al 1991 see page 22 for ordering 6xHis tag See previous page Bovine growth hormone BGH reverse priming site Permits sequencing through the insert from the 3 end BGH polyadenylation signal Efficient transcription termination and polyadenylation of mRNA Goodwin and Rottman 1992
30. tline Experimental Outline Use the following outline to clone and express your genes of interest in pBudCE4 1 Step Action Page 1 Determine a cloning strategy 3 5 2 Ligate your inserts into the vector and transform into E coli 6 20 Select transformants on Low Salt LB containing 25 50 pg mL Zeocin 3 Analyze your transformants for the presence of both inserts by 6 restriction digestion 4 Select a transformant with the correct restriction pattern and 6 sequence to confirm that both genes are cloned in frame with the C terminal peptide if desired 5 Transfect your construct into the cell line of choice 7 6 Test for expression of your recombinant proteins by western blot 8 9 analysis or functional assay For antibodies to the myc epitope the V5 epitope or the C terminal polyhistidine tag see page 22 7 Purify your recombinant proteins using a metal chelating resin 9 such as ProBond see page 21 for ordering information 8 Generate a stable cell line if desired 10 11 Methods Cloning into pBudCE4 1 General Molecular Biology Techniques E coli Strain Important Transformation Method Maintaining pBudCE4 1 Cloning Considerations For help with DNA ligations E coli transformations restriction enzyme analysis purification of single stranded DNA DNA sequencing and DNA biochemistry refer to Molecular Cloning A Laboratory Manual
31. ug mL Zeocin are sufficient to kill the untransfected host cell line Test a range of concentrations see below to ensure that you determine the minimum concentration necessary for your cell line 1 Seed cells 2 x 10 cells 60 mm plate for each time point and allow cells to adhere overnight 2 The next day substitute culture medium with medium containing varying concentrations of Zeocin e g 0 50 125 250 500 750 and 1000 ng mL 3 Replenish the selective medium every 3 4 days and observe the percentage of surviving cells 4 Observe the cells at regular intervals to determine the appropriate concentration of Zeocin that prevents growth 5 Select the concentration that kills cells in 7 10 days Continued on next page Creating Stable Cell Lines Continued Possible Sites for Linearization To obtain stable transfectants you may choose to linearize your vector before transfection While linearizing your vector may not improve the efficiency of transfection it increases the chances that the vector does not integrate in a way that disrupts the gene of interest or elements necessary for expression of the gene The table below lists unique sites that may be used to linearize your construct prior to transformation Other restriction sites are possible Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector Enzyme Restriction Site
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