Antibiotic Resistance: Ampicillin Bacterial Backbone: pbluescriptksii(+), Agilent Technologies

Transcription

1 G0656 pfiv3.2rsvmcs Coordinates Plasmid Features CMV/5 LTR hybrid Partial Gag Central Polypurine Tract RSV Promoter Multiple Cloning Sites WPRE RRE SIN 3 LTR puc origin of replication Ampicillin Resistance Marker pf1(+) phage origin Antibiotic Resistance: Ampicillin Bacterial Backbone: pbluescriptksii(+), Agilent Technologies Note: Plasmid can be analyzed by restriction endonuclease cleavage pattern by digesting with BsaI or SspI

2 Multiple cloning sites 5 to 3 G0656 pfiv3.2rsvmcs GAGCTTGCATGCCTGCAGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAA CGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCA TTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACA TGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGAT GCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTA ACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAACCAGT GCTTTGTGAAACTTCGAGGAGTCTCTTTGTTGAGGACTTTTGAGTTCTCCCTTGAGGCTCCCACAGA TACAATAAATATTTGAGATTGAACCCTGTCGAGTATCTGTGTAATCTTTTTTACCTGTGAGGTCTCG GAATCCGGGCCGAGAACTTCGCAGTTGGCGCCCGAACAGGGACTTGATTGAGAGTGATTGAGGAA GTGAAGCTAGAGCAATAGAAAGCTGTTAAGCAGAACTCCTGCTGACCTAAATAGGGAAGCAGTAGCA GACGCTGCTAACAGTGAGTATCTCTAGTGAAGCAGACTCGAGCTCATAATCAAGTCATTGTTTAAAG GCCCAGATAAATTACATCTGGTGACTCTTCGCGGACCTTCAAGCCAGGAGATTCGCCGAGGGACAG TCAACAAGAAAGGAGAGATTCTACAGCAACTAGGGGAATGGACAGGGGCGAGATTGGAAAATGGC CATTAAGAGATGTAGTAATGTTGCTGTAGGAGTAGGGGGGAAGAGTAAAAAATTTGGAGAAGGGA ATTTCAGATGGGCCATTAGAATGGCTAATGTATCTACAGGACGAGAACCTGGTGATATACCAGAG ACTTTAGATCAACTAAGGTTGGTTATTTGCGATTTACAAGAAAGAAGAGAAAAATTTGGATCTAGC AAAGAAATTGATATGGCAATTGTGACATTAAAAGTCTTTGCGGTAGCAGGACTTTTAAATATGACG GTGTCTACTGCTGCTGCAGCTGAAAATATGTATTCTCAAATGGGATTAGACACTAGGCCATCTATG AAAGAAGCAGGTGGAAAAGAGGAAGGCCCTCCACAGGCATATCCTATTCAAACAGTAAATGGAGT ACCACAATATGTAGCACTTGACCCAAAAATGGTGTCCATTTTTATGGAAAAGGCAAGAGAAGGACT AGGAGGTGAGGAAGTTCAACGCGGCCGAGTCTCAATTTTAAAAGAAGAGGTAGGATAGGAGGGAT GGCCCCTTATGAATTATTAGCACAACAAGAATCCTTAAGAATACAAGATTATTTTTCTGCAATACCA CAAAAATTGCAAGCACAGTGGATTTATTATAAAGATCAAAAAGATAAGAAATGGAAAGGACCAAT GAGAGTAGCGGCCGCATGCTAGTAGATCTGCGATGTACGGGCCAGATATACGCGTATCTGAGGGG ACTAGGGTGTGTTTAGGCGAAAAGCGGGGCTTCGGTTGTACGCGGTTAGGAGTCCCCTCAGGATA TAGTAGTTTCGCTTTTGCATAGGGAGGGGGAAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAA CATGGTAACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATTGG TGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGACATGGATTGGA CGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCTAGCTCGATACAATAAACGCC ATTTGACCATTCACCACATTGGTGTGCACCTCCAAGCTGGTACCTCGAGAAGGGCGAATTCTGCAG ATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGCTTAAGCTTAGGATCCATCCCGGGTG ATATCTAACGCGTTTTCTAGATTACTAGTTTGTCGACTTGAATTCTTATCGATTTACGCGTAAGATCT TTCTAGTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTC CTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTC

3 ATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGC AACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACC TGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCC TGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGG GAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTT CTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGC GGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCaGATcTCCCTTTGGGCCGCCGCCTCCC CGCATACCGGCAAGAAATACAACCACAAATGGAATTGAGGAGAAATGGTAGGCAATGTGGCATGTC TGAAAAAGAGGAGGAATGATGAAGTATCTCAGACTTATTTTATAAGGGAGATACTGTGCTGAGTTC TTCCCTTTGAGGAAGGTATGTCATATGAATCCATTTCGAATCAAATCAAACTAATAAAGTATGTATTG TAAGGTAAAAGGAAAAGACAAAGAAGAAGAAGAAAGAAGAAAGCCTTCAAGAGGATGATGACAGAGT TAGAAGATCGCTTCAGGAAGCTATTTGGCACGACTTCTACAACGGGAGACAGCACAGTAGATTCTGA AGATGAACCTCCTAAAAAAGAAAAAAGGGTGGACTGGGATGAGTACTGGAACCCTGAAGAAATAGA AAGAATGCGAGTATATAACCAGTGCTTTGTGAAACTTCGAGGAGTCTCTTTGTTGAGGACTTTTGA GTTCTCCCTTGAGGCTCCCACAGATACAATAAATATTTGAGATTGAACCCTGTCGAGTATCTGTGTA ATCTTTTTTACCTGTGAGGTCTCGGAATCCGGGCCGAGAACTTCGCAGGTACCCAGCTTTTGTTCC CTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTA TCCGCTCACAATTCCACACAACATACGAGCCGGGAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGA GTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCC AGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTT CCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGC AAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGAC GAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACC TGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGC GCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG GCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTT CGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGT TTGCAAACAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATC TTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGT CTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCAT AGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTG CTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC GGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGC ATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGC GGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTA AAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTT CTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAAT GTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGTGCACATTTCCCCGAAAAGTG CCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTT TAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGT GTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAA AACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAG CCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCA AGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCG CGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTAT TACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCC AGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGG AGCTCCACCGCGG

4 Plasmid Analysis by Restriction Endonuclease Cleavage Pattern pfiv3.2rsvmcs GTVC ID#: G0656 Plasmid Prep Date: 5/23/08 Concentration: 2.9ug/ul Predicted fragments size: BsaI-HF SspI-HF 2731 bp 2731 bp 2179 bp 2176 bp 1376 bp 1249 bp 130 bp

5 Information and Cloning Suggestions for Working with pfiv3.2 plasmids: Background: Unlike many other retroviruses, lentiviral vectors have the advantage of infecting both dividing and non-dividing cells. However, they retain stable and long-term expression which is heritable. Lentiviral vectors are produced through a triple plasmid transfection reaction, which includes a transgene shuttle plasmid, a packaging plasmid, and an envelope plasmid. Lentivirus can be pseudo-typed with different envelopes to infect certain tissues and cell lines with greater efficiency. These viral vectors are able to accommodate transgene inserts of approximately 6.5Kb. Viral titers range between 1.0E+7 to 5.0E+8 TU/ml. The Feline Immunodeficiency viral vectors are replicationincompetent and have not been found to infect humans. There are several lentiviral reporter vectors available for purchase from the Viral Vector Core. FIVbased lentiviral vectors are available to University of Iowa investigators and outside users. Investigators outside the University of Iowa may obtain these vectors through a three party Material Transfer Agreement with Novartis Corporation and the University of Iowa. pvetlcmvmcs Backbone Origin: This cloning and expression plasmid was developed by Chiron Technologies: A series of stepwise PCR amplifications were inserted in the pbluescriptksii(+) phagemid to create the FIV backbone plasmid called ptfivl. This plasmid contained the complete 5 LTR and 3 LTR and a long region (0.55kb) of the FIV gag gene. This backbone was further modified to replace the U3 region of the 5 LTR with the CMV promoter-enhancer to create the ptc/fl backbone. The CMV promoter was then again inserted at the 5 end but immediately before the multiple cloning sites and the backbone was renamed pvet L CMVskh10 (currently named pvet L CMVmcs). FIV3.2 Plasmid Vector Origin: The FIV3.2 plasmid is a derivative of Novartis pvet L CMVmcs cloning plasmid (description above). Several modifications were made to the original construct to improve safety and transgene expression. This plasmid was modified by Patrick L. Sinn, PhD (University of Iowa): The lentiviral central polypurine tract and the central termination sequence were added directly downstream of the gag sequence. The Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element, WPRE, was added directly upstream of the rev response element, RRE. The major splice donor and the initial gag sequence each have a mutation but the packaging signal was retained. The FIV 3 LTR was rendered self-inactivating by deleting a portion of the U3 region. Insert Size: Lentiviral vectors are able to accommodate transgene inserts of approximately 6.5Kb. Typically, vector titers decrease inversely proportionally with increasing transgene sizes above 6.5Kb. Note: When cloning your gene of interest, do not include a polyadenylation signal. Inclusion of a poly A signal will not allow proper packaging of lentiviral particles.

6 Lentiviral Production: The University of Iowa Viral Vector Core utilizes a triple plasmid transfection system: a packaging plasmid, envelope plasmid and transgene plasmid. The packaging plasmid encodes for the necessary viral proteins in trans, but they are not packaged into the particles. The envelope plasmid encodes for the envelope protein in trans. The transgene plasmid contains your gene of interest. It also retains the minimal cis acting viral sequences necessary for packaging, reverse transcription and integration. So, when the virus particles are assembled, the accessory genes required for subsequent viral replications are not included, therefore, making the vector replication deficient. We will require 250ug of purified transgene plasmid for production of your vector for a single 500ul prep of concentrated FIV vector. The packaging and envelope plasmids are provided by the UI VVC. The services provided for production are: Triple plasmid transfection Viral particle concentration Titer Assay (transducing units/ml) Plasmid Amplification and QC: Amplification of the pfiv3.2 plasmids: The pfiv3.2 plasmid backbone is ampicillin resistant. Pick a single colony and inoculate in LB broth containing 100g/ml of Ampicillin. Grow on LB agar plates containing 100ug/ml of Ampicillin. We recommend transforming your final miniprep into a stable competent cell line such as SURE2, Stbl2, or Stbl3 to avoid unwanted recombination in viral vector plasmids. We prefer Stbl3 in the Viral Vector Core. Sub-cloning, however, can be difficult in these competent cells. We recommend DH5a competent cells for subcloning. Transform your final, confirmed miniprep into a stable competent cell line. Maxi preps: Use high quality, endotoxin free plasmid purification kits for DNA extractions. The Viral Vector Core also provides maxi prep service for viral vector production. Restriction Digest Analysis: In addition to sequencing, pfiv3.2 plasmids can be digested with BsaI or SspI to confirm the integrity of the plasmid. It is recommended to run a sample of the un-cut and linearized plasmid at the same time. The Viral Vector Core requires a plasmid digest along in addition to the sequence and a map for each plasmid submission. Sequencing Primers for Verification of the Transgene Insert: Additional primers will need to be designed specifically for each cloning project to confirm insertion of your gene of interest. WPREreverse 5 - AGCTGACAGGTGGTGGCAAT-3 Protein expression or mirna knockdown All the plasmids provided at the Viral Vector Core are also expression vectors. Prior to sending the plasmid for virus production, we recommend testing for protein of interest expression or mirna knockdown of protein expression. References: Johnston JC, Gasmi M, Lim LE, Elder JH, Yee JK, Jolly DJ, Campbell KP, Davidson BL, Sauter SL. Minimum Requirements for Efficient Transduction of Dividing and Nondividing Cells by Feline Immunodeficiency Virus Vectors. J Virol June; 73(6): Stein CS, Davidson BL. Gene Transfer to the Brain Using Feline Immunodeficiency Virus-Based Lentivirus Vectors. Methods Enzymology 346: , Harper SQ, Davidson BL. Plasmid-based RNA interference: Construction of small-hairpin RNA (shrna) expression vectors. Methods Mol Biol 309: , 2005.

7 Harper SQ, Staber PD, Beck CR, Fineberg SK, Stein CS, Ochoa D, Davidson BL. Optimization of Feline Immunodeficiency Virus Vectors for RNA Interference. J Virol 80(19): , Boudreau RL, Davidson BL. Chapter 14 - Generation of Hairpin-Based RNAi Vectors for Biological and Therapeutic Application. Methods Enzymology 507: , Please contact us with any questions: Viral Vector Core vectors@uiowa.edu University of Iowa 500 Newton Road 221 Eckstein Medical Research Building Iowa City, IA Tel: (319)