G0752 pfiv3.2mcswtiresegfp

Transcription

1 G0752 pfiv3.2mcswtiresegfp Plasmid Features Coordinates Feature CMV/5 LTR Hybrid Partial Gag Central Polypurine Tract Multiple Cloning Sites wtires egfp WPRE Rev Response Element 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 G0752 pfiv3.2mcswtiresegfp GAGCTTGCATGCCTGCAGGTCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGAC CCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTC AATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCC CCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT CCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAA TGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTG TTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCG GTAGGCGTGTACGGTGGGAGGTCTATATAACCAGTGCTTTGTGAAACTTCGAGGAGTCTCTTTGTTGAGG ACTTTTGAGTTCTCCCTTGAGGCTCCCACAGATACAATAAATATTTGAGATTGAACCCTGTCGAGTATCTG TGTAATCTTTTTTACCTGTGAGGTCTCGGAATCCGGGCCGAGAACTTCGCAGTTGGCGCCCGAACAGGGA CTTGATTGAGAGTGATTGAGGAAGTGAAGCTAGAGCAATAGAAAGCTGTTAAGCAGAACTCCTGCTGACCT AAATAGGGAAGCAGTAGCAGACGCTGCTAACAGTGAGTATCTCTAGTGAAGCAGACTCGAGCTCATAATCA AGTCATTGTTTAAAGGCCCAGATAAATTACATCTGGTGACTCTTCGCGGACCTTCAAGCCAGGAGATTCGC CGAGGGACAGTCAACAAGAAAGGAGAGATTCTACAGCAACTAGGGGAATGGACAGGGGCGAGATTGGA AAATGGCCATTAAGAGATGTAGTAATGTTGCTGTAGGAGTAGGGGGGAAGAGTAAAAAATTTGGAGAAG GGAATTTCAGATGGGCCATTAGAATGGCTAATGTATCTACAGGACGAGAACCTGGTGATATACCAGAGA CTTTAGATCAACTAAGGTTGGTTATTTGCGATTTACAAGAAAGAAGAGAAAAATTTGGATCTAGCAAAGA AATTGATATGGCAATTGTGACATTAAAAGTCTTTGCGGTAGCAGGACTTTTAAATATGACGGTGTCTACTG CTGCTGCAGCTGAAAATATGTATTCTCAAATGGGATTAGACACTAGGCCATCTATGAAAGAAGCAGGTG GAAAAGAGGAAGGCCCTCCACAGGCATATCCTATTCAAACAGTAAATGGAGTACCACAATATGTAGCAC TTGACCCAAAAATGGTGTCCATTTTTATGGAAAAGGCAAGAGAAGGACTAGGAGGTGAGGAAGTTCAAC GCGGCCGAGTCTCAATTTTAAAAGAAGAGGTAGGATAGGAGGGATGGCCCCTTATGAATTATTAGCACAA CAAGAATCCTTAAGAATACAAGATTATTTTTCTGCAATACCACAAAAATTGCAAGCACAGTGGATTTATTA TAAAGATCAAAAAGATAAGAAATGGAAAGGACCAATGAGAGTAGCGGCCGCATGCTAGCTTAAGCTTAG GATCCATCCCGGGTGATATCTAACGCGTTTTCTAGATTACTAGTTTGTCGACTTGAATTCGCCCCTCTCCC TCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTAT TTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCAT TCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCT CTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGC GACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTG CCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCT GAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTACACATGCTTTACATGT GTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACAC GATGATAATATGGCCACAACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCT GGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTC

3 GTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTC TTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTAC AAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGA CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATAT CATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCA GCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGAC AACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCT GCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAATCGATTTACG CGTAAGATCTTTCTAGTAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATG TTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTT TCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAA CGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAG CTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCC GCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCT TTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGC CCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTT CGCCCTCAGACGAGTCAGATCTCCCTTTGGGCCGCCGCCTCCCCGCATACCGGCAAGAAATACAACCACA AATGGAATTGAGGAGAAATGGTAGGCAATGTGGCATGTCTGAAAAAGAGGAGGAATGATGAAGTATCTC AGACTTATTTTATAAGGGAGATACTGTGCTGAGTTCTTCCCTTTGAGGAAGGTATGTCATATGAATCCATT TCGAATCAAATCAAACTAATAAAGTATGTATTGTAAGGTAAAAGGAAAAGACAAAGAAGAAGAAGAAAGAAG AAAGCCTTCAAGAGGATGATGACAGAGTTAGAAGATCGCTTCAGGAAGCTATTTGGCACGACTTCTACAAC GGGAGACAGCACAGTAGATTCTGAAGATGAACCTCCTAAAAAAGAAAAAAGGGTGGACTGGGATGAGTAC TGGAACCCTGAAGAAATAGAAAGAATGCGAGTATATAACCAGTGCTTTGTGAAACTTCGAGGAGTCTCTT TGTTGAGGACTTTTGAGTTCTCCCTTGAGGCTCCCACAGATACAATAAATATTTGAGATTGAACCCTGTCG AGTATCTGTGTAATCTTTTTTACCTGTGAGGTCTCGGAATCCGGGCCGAGAACTTCGCAGGTACCCAGCT TTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATT GTTATCCGCTCACAATTCCACACAACATACGAGCCGGGAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGA GTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCT GCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTC ACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGT TATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGAC GCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCC TCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT GGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCG GTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCG CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTG GTAGCGGTGGTTTTTTTGTTTGCAAACAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT CAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAA ACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATC CATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCT GCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCT AGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCAC GCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCAT GTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTA TCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGAC TGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCA ATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGC GAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATC TTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAG GGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAG GGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGTGCACAT

4 TTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCA GCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGT TGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAA AAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTG CCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGA ACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGT CACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATT CAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGG GGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACG GCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGAGCTCCACCGCGG Plasmid Analysis by Restriction Endonuclease Cleavage Pattern pfiv3.2mcswtiresegfp VVC ID#: G0752 Plasmid Prep Date: 3/15/13 Concentration: 1.5ug/ul Predicted Fragments BsaI SspI EcoRI + ClaI 3560bp 3560bp 5801bp 2179bp 2176bp 1314bp 1376bp 1249bp 130bp

5 Encephalomyocarditis Virus Internal Ribosomal Entry Site (EMCV IRES): The VVC IRES plasmids were changed in 2013 to replace an attenuated EMCV IRES with the native wild type EMCV IRES for improved expression. There were two changes made to the attenuated IRES sequence to convert it back to the native IRES sequence. First, a single base pair was deleted in the A7 bifurcation loop to th change it back to the native A6 loop. Second, the IRES sequence was extended to include the native 11 and th 12 ATGs.Translational efficiency of EMCV IRES in bicistronic vectors is dependent both upon the IRES sequence and gene location. Bochkov YA, Palmenberg AC Testing a particular sequence for IRES activity relies on a bicistronic reporter construct. An IRES segment drives translation of the downstream protein coding region best when it is located between two open reading frames. Without an upstream open reading frame, the expression is present but weak. Calcium Phosphate Transfections pfiv3.2mcswtiresegfp pfiv3.2cmvmcswtiresegfp pfiv3.2cmvmcherrywtiresegfp Note: cells transfected are the 293FT cell line from Invitrogen catalog #R Lentiviral Production:

6

7 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 nondividing 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 replication-incompetent and have not been found to infect humans. There are several lentiviral reporter vectors available for purchase from the Viral Vector Core. FIV-based 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 promoterenhancer 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.

8 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. 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

9 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: , 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)