RAPAd® miRNA Adenoviral Expression System
Contents
1. GCATATAAATA CTGCATATAAATTGTAAC AAGAG G CATATTGCTAA G Cc GCAATAG G C TTTGC GAATTCTGACACACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGT AGCAGCTACAATCCAGCTACCATTCTGC AY ATGGTTGGGATAAGGCTGGATTATTCTGAGTCCAAGCTAGGCC AATCATGTTCATACCTCTTATCTTCCTCCCACAGCTCCTGGGCAACGTGCTGGTCTGTGTGCTGGCCCATCAC GGCAAAGCACGTGAGATCT GCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCG GA TGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCAC CCTGACC ATCTTCF TACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACC TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGT T CGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGG AGGACGGCAACAT CCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCAT CAAGGTGAACTTCAA GATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCT GCTGCCCGACAACCACTAC CTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACG AGCTGTACAAGGCCACCATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGA CTACCCCGCCACGCGCCACACCGT CGATCCGGACCGCCACATCGAGCGGGT CACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAG GTGTGGG TGAGCGG AACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACG2. es G GGGCGCGGGGCAT GGCC CGACGCTCAAGTCAGAGG CCCCATTATGA CAAGGCCAGCAAAAGGCCAGGAACCGT GGCGAAACCCGACAGGACTAT CTTCTCGCTTC ACCGGATACCTGTCCGCCTTTCTCCC TAAAAAGGCCGCG TAAAGATACCAGGCGTTTCCCCCTGGAAG GACTATCG AY GGGGCGGC CAGCGTAGTCTGGGTCACGGTGAAGGGGT CGCCCTGCGCGTCGGCCAGGTAGCATTTGACCATGGTG CCGCGGCGTGGCCCTTGGCGCGCAGCTTGCCCTTGGAGGAGGCGCCGCACGAGGGGCAGTGCAGACT CCGGGGAGTAGGCAT CCGCGCCGCAGGCCCCGCAGACGGTCTCGCA TGCTTTTTGATGCGTTTCT CGACCGATGCCCTTGAGAGCCTTCAACCCAG GCAACTCGTAGGACAGGTGCCGGCAGCGCTC CGCCGCACT CGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGC CGGCGAGAAGCAGGCCA CCATAATGATGGCAATGGGCCCACGGGCGGCGGCC TTTACAAAGCGCGGGCGGAGGGTGCCAGACTGCG GTCTACCTGCGGGGCGAT ACCGCAGCCGGTGGGCCCGTAAATCACACCTATT CTGACTCGCATGTTTTCCCTGACCA GAGCG CGCTG GCGCTCC GAGGGCGTAGAGCTTGG GGCCGTTCGGGGTCAAA CCGTGTCCCCGTATACA CGCCGGCAT ATGACT TGGCGGC CGGCGGCATCGGGATGCCCGCGTTG CGGGAAGCGTGGC GGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT CCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTG GGTATCTGCGCTCTGCTGAAGCCAGTTACC GCTTTCTCA CGGAAAAAGAGT GGTCATGAGATTA GCTTAA CAGTGAGGCACCTATCT GG GT GCCATTGCTGCAGGCATCGTGGTGTCACGCTCG TCAAGGCGAGTTACATGA TGGTTATGGCAGCACTGCA GCGGCGACCGAGTTGCTC AAACTCTCAAGGAT
3. Product Manual RAPAd miRNA Adenoviral Expression System Catalog Number VPK 253 1 kit FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction MicroRNAs miRNAs are 18 24 nucleotide RNA molecules that regulate the stability or translational efficiency of target mRNAs These regulatory RNAs function by acting as sequence specific guides which recruit a large protein complex known as the RNA induced silencing complex RISC to target mRNAs which are subsequently silenced Diverse functions have been attributed to miRNAs including the regulation of cellular differentiation proliferation and apoptosis Moreover significant evidence has accumulated implicating a fundamental role for miRNAs in the development of cancer miRNAs are initially transcribed as long precursor transcripts known as primary microRNAs pri miRNAs Within these transcripts the mature miRNA sequences are found in 60 80 nucleotide hairpin structures Mature miRNAs are generated from pri miRNAs by sequential processing Figure 1 Pri miRNAs are initially recognized in the nucleus by the microprocessor complex which includes as core components the RNase III enzyme Drosha and its obligate partner DGCR8 This complex excises the hairpin structure containing the mature miRNA sequence The liberated hairpins referred to as precursor miRNAs pre miRNAs are recognized by the nuclear ex
4. 353 of Ad5 EF 1o Promoter human B globin intron BamHI Nhel GFP Puro Fusion GFP 1355 2071 Puro 2078 2677 SV40 pA 3328 5792 of Ad5 B Lactamase pacAd5 miR GFP Puro Shuttle Vector Sequence AATTAAT GGGCGGG TGTACACAGGAAGT AGAGGAA CCAGAA AAGCAAGCATCATCAATAATATACCTTATTTTGGATTGAAGCCAATATGATAATGAGGGGGTGGAG TGACGTAGTAGTGTGGCGGAAGTGTGATGTTGCAAGTGTGGCGGAACACATGTAAGCGACGGATGTGGCAAAAGTGACGTTTTTGGTGTGCGCCGG TGACAATTTTCGCGCGGTTTTAGGCGGATGTTGTAGTAAA GGGCGTAACCGAGTAAGAT GTGAAATCTGAATAATTTTGTGTTACTCATAGCGCGTAATATTTGTCTAGGGAGATCCGGTACCGTT1 CACAGGTACACATA TTGTGACGTGGCGCGGGGCGTGGGAACG TTGGCCATTTTCGCGGGAAAACTGAATA TAAACTCGAGGTCGACGGTATCGAT CCCCGTCACCACCCCCCCCAACCCGCCCCGACCGGAGCTGAGAGTAATT CATACAAAAGGACTCGCCCCTGCCTTGGGGAATCCCAGGGACCGTCGTTAAACT CCCACTAACGTAGAACCCAGAGAT CGCTGCGTTCCCGCCCCCTCACCCGCCCGCTCTCGTCATCACTGAGGTGGAGAAGAGCATGCGTGAGGCTCCGGTGCCC GTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGT CGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGA AAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC CGCAACGGG TGCC GACCAAATCAGGGTAATTTTGCATTTGTAA AAAAAATGC AATAC CCCTAATCTCT CTTCT TTAATATAC TGTTTATC ATT CTTTCAGGGCAATAATGATACAATGTATCATGCCTC GCACCA TCTAAAGAATAACAGTGATAATTTCTGGG AAG CCGATTAGTTCTCGAGGATCCGACTGAAGTCGCTAGCTCGAGC GGAGAATATTTC
5. without written permission from Cell Biolabs The patented RAPAd technology is covered by a license from University of Iowa By the use of this product you accept the terms and conditions of all applicable Limited Use Label Licenses You may contact our Business Development department at busdev cellbiolabs com for information on sublicensing this technology Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2009 2018 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing 12 eo CELL BIOLABS INC A a A
6. CCGC GCGTCAGAATGTGATGGGCTCCAG TGGAGAC CGCCCGCGGGATTGTGACTGACTTTGC CCTGAGCCCGCTTGCAAGCAGTGCAGCTTCCCGTTCAT CAATTGGATTCTTTGACCCGGGAACTTAATGTCGTTTCTCAGCAGCTGTTGGATCTGCGCCAGCAGGT TTTAAAACATAAATAAAAAACCAGACTCTGTTTGGATTTGGAT CAAGCAAGTGTCTTGC GCGGTCTCGGTCGTTGAGGGTCCTGTGTATTTTTTCCAGGACGTGGTAAAGGTGACTCTGGATGTTCAGATACATGGGCATAAGCCCGTCTC TAGCACCACTGCAGAGCTTCATGCTGCGGGGTGGTGTTGTAGATGATCCAGTCGTAGCAGGAGCGCTGGGCGTGGTGCCTAAAAATGTC TGATTGCCAGGGGCAGGCCCTTGGTGTAAGTGTTTACAAAGCGGTTAAGCTGGGATGGG GTTGGCTATGTTCCCAGCCATATCCCTCCGGGGATTCATGTTGTGCAGAACCACCAGCACAGTGTATCCGGTGCACTTGGGAAATTTGTCA GCAGCCTCCGCCGCCGCTTCAGCC gt CCGCCCGCGATGACAAGTTGACGG TTCTGCCCTGAAGGCTTCCTCCCC GTCTTTA TTAGGGGT GCGCGCGCGGTAG 10 TGCATACGT GGGGATATGAGATGCATCTTGGAC N CELL BIOLABS INC CAT GCTG CTCT TCCC GCCC TGGG CAGT TGTA GTAG CTTAGAAGGAAATGCGTGGAAGAACTTGGAGACGCCCTTGTGACC TGGGCGAAGATAT GGTTCCAT GAAGAAAACGGTTI GTATAA CTGGGATCAC ACGGCAG TCATAG GAC AGTCGGTGC GTCC GTCTTC TTGCGG ATAGGC CTCCCTCGTGCGC TATCTCAGTTCGG ACG G cc GCCTAAC ACGGC ACCACCGCTGGTAGCGG CTCAGTGGAACGAAAACT CTAAAGTATATATGAG CTCCCC
7. CTTACCGCTGT GAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAA TCAGGGTTATT CCCCCATGT GTGCAAAAAAGCGGTTAGCTCCTTCGG Cc CCTGCAACTTTATCCGCCTCCATCCAG TGGTAGCTC CAAGAAGATCCT TTGATC TAAATTAAAAA CAGCGATCTGTC TACCGCGAGACCCACGCTCACCGGC AY CTATTAAT GGTATGGC FCG CCTCCGAT AATTCTC ACTGTCATGCCATCCGTAAGATGCTTT GCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACT CGTTGTCAGAAG CTGTGACT GG GAGTACTCAACCAAGTCA G TGCTGGCG CTACGGGG GAAGTTT TCGTTCATCCATAGT TTCC AGCTCACGCTGTAGG TAACTATCGTC TGATCCGGCAAACAA GAG CTGACG AAA CAA TGCCTGA CCAGAT GCCGGGAAGCTAGAGT CATTCAGCTCCGGT1 AAGTTGGCCGCAGTGTTAT Te GAGA GT CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA CCAGTTCGATGTAACCCACTCGTGCACCCAACTGA TAAAAGTGCTCA CA CTTCAGCATC G GAATAC TGGAAAACGTTCTI ACTTTCACCAGCGTT TATCAG TAAGTAG CCCAACGA TCACTCA GAGAATAGTGTAT CGGGGCGA CATACTCTTCC TAAGAAACCA TATTATCATGACATTAACC References 1 2 3 AGGGGT TTTCAATAT CCGCGCACAT ATAAAAATAGGCGTATCACGAGGCCCTTTCGTCT TCAAGAA CCCCGAAAAG CTGGGT ATTGAAGCATTTA GCCACC microRNA sequences listed in Sanger s miRBase http microrn
8. GTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT CAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAG TTCGCCAGTTAATAGTTTGCGCAACGT ACAC GGTTTTT AAACTTGG AACGTCA gt CCGGCCCAGGGGCGTAGT CCGGGGTAGGGGAGATCAGCTGGGAAGAAAGCAGGTTCCTGAGCAGCTGCGAC ACCGGGTGCAACTGGTAGTTAAGAGAGCTGCAGC AATCCGCCAGAAGGCGCT TTGACCAAGCAGTT gt CGCCGCCCAGCGATAGCAGTTCTTGCAAGGAAGCAAAGT CCAGGCGGTCCCACAGCTCGG CGTCCAGACGGGCCAGGGTCATGTCT GGGCTGCGCGCTGGCCAGGGTGCGCTTGAGGCTGG CCAGCCCCT GCGCGAGAAATACCGA AACCAGGTTTCCCCCAT GAGAGGCC TATCA ATTCGGAA CGACGCGCTGGGC GCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACG CTTGCTGGCGTTCGCGACGCGAGGCTGGA CAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGC CCGCCCCCCTGACGAGCATCACAAAAA GTTCCGACCCTGCCGC AGGTCGTTCGCTCCAAGCT AGAAGGACAGTATT TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGA TCACGTTAAGGGATTT CAAAAAGGATCTTCACCTAGATCCTT CTGACAGTTACCAA CCAAGA TCCATGCA TCG AGTTGTGTTCCAGGATGAGATCG CATAGGCCA ACCCTCACAGATTTGCATTTCCCACGCTT GAG GCCGTCATCCCTGAGCAGGGGGGCCACT CG AAGCATGTCC TTTCAACGG TGAGACCGT CACCTGCTCTACGGCA CTCGATCCAGCA CAGATGGGGGGATCAT CCGCCGTAGGCATGCT ATCTCCTCGTTT CCACGGGCGCAGGGTCC CG CCTGCTGGTGCTGAAGCGCTGCCGGTC CGCGGG ACCTCTGG CCA CAGC GGGTCA CCTTCCGG 4 CCACGAGCCAGGTGAGC GAGCCGGTGTCCACGCTCGGTGACGAAAAGGC
9. PAd Adenoviral Expression System provides a much faster and safer method to generate RCA free recombinant adenovirus at high titer The RAPAd system uses a novel Ad backbone devoid of the left hand ITR the packaging signal and E1 sequences There is no need to perform the bacterial in vitro homologous recombination pAdEasy method and also the multiple plaque isolations standard homologous recombination method in packaging cell line The RAPAd system allows for generation of a recombinant virus within 2 weeks and the virus produced contained virtually no contaminating Ela sequences or replication competent virus RCA Cell Biolabs RAPAd miRNA Adenoviral Expression System is designed to rapidly produce recombinant adenovirus that expresses an individual miRNA precursor in its native context while preserving putative hairpin structures to ensure biologically relevant interactions with endogenous processing machinery and regulatory partners leading to properly cleaved microRNAs Individual miRNA precursor from any species can be cloned between BamHI and Nhe I sites Figure 2 RAPAd miRNA Adenoviral Expression System contains the following unique features e miRNA Processing miRNA stem loop precursor in its native context is cloned between BamHI and Nhe I sites To preserve the putative hairpin structure and proper endogenous processing miRNA stem loop sequence is flanked by its native intron sequence e EF la Promoter ensures a h
10. T GTGATGC GGTT GCAGCTC TCGCGGACGACGGCGCCGCGGTGGCGGTCT ATGAGG AGGAACCAGCCTGTGATGCTGGATGTGACCGAGGAGCTGAGGCCCGATCACTTGGTGCTGGCCTGCACCCGCGCT ATTGAGG GGACCACGCCGGAGAGCGT CGAAGCGGGGGCGGTGTT CGCCGAGATCGGCCCGCGCATGGCCGAGT TTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGC CCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCC CGACCACCAGGGCAAGGGT CTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGC CGAGTGCCCGAAGGACCGCGCGACCTGGTGCATGACCCGCAAGCCCGGTGCCTGAG CGGCCGCCACCGCGGGGAGAT CCAGACATGATAAGATACATTGATGAGTT TGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTT ATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTG AAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCCGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTTGACACAT CCGGAGACGGTCACAGCT TGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCC CGACTCTAGTCCCCGCGGTGGCAGAT CTGGAAGGTGCTGAGGTACGATGAGACCCGCACCAGGTGCAGACCCTGCGAGTGTGGCGGTAAACATATT TGAGTTTGGCTCTAGCGATGAAGAT TACTGAAATGTGTGGGCGTGGCTTAAGGGTGGGAAAGAATATATAAGGTGGGGGTCTTA GTAGTTT CATGAGCACCAACTCG GATGGAAGCATTGTGAGCTCATATTTGACAACGCGCATGCCCCCATGGGCCGGGG1 GATGGTC CAGCCAC TTTGGCA AATGCGG GGGACCA GTGGAGG AGCAAGC TTTTTAG GCCCCGTCCTGCCCGCAAACTCTACTACCTTGACCTACGAGACCGTGTCTGGAACGCCG GTATCTGT GCAGCAGCCGCCG GGGA ACAG
11. a sanger ac uk sequences Homologous recombination in packaging cell line 95 2509 14 pAdEasy System RAPAd System Recent Product Citations 1 John B C Sander and D S Marks 2006 Methods Mol Biol 342 101 13 Bett AJ Haddara W Prevec L and Graham FL 1994 Proc Natl Acad Sci U S A 91 8802 6 pancreatic beta and alpha cells J Biol Chem doi 10 1074 jbc M115 650705 2 suppresses the dissemination of ovarian cancer cells Mol Cancer Ther 13 2081 2091 inhibits PDGF BB induced cell proliferation and migration through targeting orphan nuclear receptor NOR1 Cardiovascular Res 10 1093 cvr cvt082 prevents neointimal formation Cardiovasc Res 95 517 526 11 gt gt GACGTC He T C Zhou S da Costa L T Yu J Kinzler K W et al 1998 Proc Natl Acad Sci USA R D Anderson R E Haskell H Xia B J Roessler and B L Davidson 2000 Gene Ther 7 1034 8 Mohan R et al 2015 Differentially expressed microRNA 483 confers distinct functions in Sugiyama K et al 2014 Expression of the miR200 family of microRNAs in mesothelial cells Li P et al 2013 MicroRNA 638 is highly expressed in human vascular smooth muscle cells and Wang Y S et al 2012 MicroRNA 195 regulates vascular smooth muscle cell phenotype and CELL BIOLABS INC ae Notice to Purchaser This product is sold for research and development purposes only and is not to be incorporated into products for resale
12. an expression vectors pEP miR or pEPGP miR are based on the following design and the resulting overexpression of the mature miRNA is confirmed by Northern blot or Real Time PCR Here we use human let 7a 2 miRNA as an example 1 Download desired miRNA stem loop sequence from Sanger s miRNA database http microrna sanger ac uk sequences Homo sapiens let 7a 2 stem loop structure uu g u uagaa ua a agg gag uag agguuguauaguu uc ee ee a ee a ucc uuc auc uccgacaugucaa ag u g c uag gg a v a gc CELL BIOLABS INC ae o gt Homo sapiens let 7a 2 stem loop sequence AGGUUGAGGUAGUAGGUUGUAUAGUUUAGAAUUACAUCAAGGGAGAUAACUGUACAGCCUCCUAGCUUUCCU 2 Blast search miRNA stem loop sequence http blast ncbi nlm nih gov Blast cgi In gt ref NT_033899 7 Hs11_ 34054 D Homo sapiens chromosome 11 genomic contig reference assembly Length 38509590 Query 1 AGGTTGAGGTAGTAGGTTGTATAGTTTAGAATTACATCAAGGGAGATAACTGTACAGCCT 60 PITT PP PI PEEP PEEP PEEP T A BEREE Sbjct 25579717 AGGTTGAGGTAGTAGGTTGTATAGTTTAGAATTACATCAAGGGAGATAACTGTACAGCCT 25579658 Query 61 CCTAGC CC 72 Sbjct 25579657 CCTAGC CC 25579646 3 PCR and Cloning 1 Add 100 base native flank sequence to both upstream and downstream of the miRNA stem loop Human let 7a 2 miRNA precursor sequence including the 100 base flank sequences on both ends of the stem loop let 7a 2 stem loop sequen
13. ce is underlined GCCCAAATAGGTGACAGCACGATGAATCATTATAAGACTAACTTGTAATTTCCCTGCTTAAGAA ATGGTAGTTTTCCAGCCATTGTGACTGCATGCTCCCAGGTTGAGGTAGTAGGTTGTATAGTTTA GAATTACATCAAGGGAGATAACTGTACAGCCTCCTAGCTTTCCTTGGGTCTTGCACTAAACAAC ATGGTGAGAACGATCATGATTCCTCCAGGCCTTTTCTCCCTATGAAAGGTAAGATTGGGTACGA TTATTTTATGGTATTT 2 Design PCR primer including BamHI site at forward primer with four extra bases and Nhel site at reverse primer Forward PCR Primer tcga ggatcc BamHI 21 nt Reverse PCR Primer tcga gctagc NheI 21 nt For human let 7a 2 miRNA precursor Forward PCR Primer tcga ggatcc gceccaaataggtgacagcacg Reverse PCR Primer tcga gcetagce aaataccataaaataatcgta 3 PCR the miRNA precursor from genomic DNA and clone into the BamHI Nhel sites of the expression vector PCR Product of let 7a 2 precursor let 7a 2 stem loop sequence is underlined 1 tcgaggatce gcccaaatag gtgacagcac gatgaatcat tataagacta acttgtaatt 61 tccctgctta agaaatggta gttttccage cattgtgact gcatgctcce aggttgaggt 7 CELL BIOLABS INC 121 agtaggttgt atagtttaga attacatcaa gggagataac tgtacagcect cctagctttc 181 cttgggtctt gcactaaaca acatggtgag aacgatcatg attcctccag gccttttctc 241 cctatgaaag gtaagattgg gtacgattat tttatggtat ttgctagctc ga 4 Validate the insert by DNA sequencing Forward Sequencing Primer TTTGCACCATTCTAAAGAAT Reverse Sequencing Primer AAACCTCTTACATCAGTTAC Preparation of Recombinant Adenovirus I Vector Linearization with PaclI 1 2 Digest a suffic
14. delines for all materials containing BSL 2 organisms Vector Features pBR322 EF 1 i uman pacAd5 amp globin miR GFP Puro lt Intron 7 9 kb BamHl Ad5 Desired miRNA Nhe Precursor SV40pA GFP Puro Multiple Cloning Sites GATTAGTTCTCGAGGATCCGACTGAAGTCGCTAGCTCGAGCTTTTGGA BamHI Nhel Figure 2 pacAd5 miR GFP Puro Shuttle Vector 7900 bp Ampicillin resistant Xho I Digestion 26 bp 660 bp 7214 bp eo CELL BIOLABS INC lt lt Pacl Pacl pacAd5 9 2 100 34 9 kb AE3 A720 bp Ad5 Figure 3 pacAd5 9 2 100 Vector 34947 bp Ampicillin resistant The novel pacAd5 9 2 100 Ad backbone vector is devoid of the left hand ITR the packaging signal and E1 sequences Pacl eGFP pacAd5 CMV eGFP 6 9 kb SV40pA Figure 4 pacAdS5 CMV GFP Control Vector 6935 bp Ampicillin resistant pacAd5 CMV GFP Features 3 10 Pacl 16 368 1 353 of Ad5 385 912 CMV Promoter 992 1711 GFP 1713 2160 SV40 pA 2161 4615 3328 5792 of Ad5 5867 6727 B Lactamase eo CELL BIOLABS INC Pacl pacAd5 CMV ntLacZ oer 9 4 kb SV40pA Figure 5 pacAdS5 CMV ntLacZ Control Vector 9278 bp Ampicillin resistant pacAd5 CMV ntLacZ Features 3 10 Pacl 16 368 1 353 of Ad5 385 912 CMV Promoter 1105 4148 ntLacZ 4193 4640 SV40 pA 4641 7095 3328 5792 of Ad5 8347 9210 B Lactamase miRNA Precursor Cloning All of our premade human amd mouse miRNA precursor clones in mammali
15. e per day under the microscope for CPE When CPE is nearly complete i e most cells rounded but not yet detached from the flask harvest cells by pipetting media up and down to wash the infected cells from the flask into the media 4 Pool infected cells and medium Pellet cells by centrifugation at 1000 g for 5 minutes Remove supernatant resuspend cell pellet in medium or in 10 mM Tris pH 8 0 100 mM NaCl 0 25 0 5 mL per T75 flask 5 Release the adenoviruses from the cell suspension with three freeze thaw cycles Centrifuge at 3000 g for 10 minutes to pellet the cell debris Discard the pellet and save supernatant as viral stock 6 The viral supernatant can be stored at 80 C or immediately purified or titered Example of Results The following figures demonstrate typical results of generating recombinant adenovirus One should use the data below for reference only This data should not be used to interpret actual results Figure 6 Generation of recombinant adenovirus using the RAPAd Adenoviral Expression System 293 cells were transfected with Pacl linearized pacAdS CMV GFP vector and pacAd5 9 2 100 vector Plates were examined for the presence of viral foci under inverted fluorescence microscope CELL BIOLABS INC aeS gt Appendix pacAd5 miR GFP Puro Shuttle Vector Features 3 10 16 368 409 804 837 1327 1053 1058 1069 1074 1355 2677 2697 2938 3120 5584 6832 7692 Pacl 1
16. ient amount of the pacAd5 shuttle vector containing gene of interest and the pacAd5 9 2 100 Ad backbone vector with Pacl Run 0 5 ug of each digested DNA and undigested DNA on a 0 8 agarose gel to confirm the completion of PacI digestion For pacAd5 9 2 100 one band of 33 kb and a second band of 2 0 kb Remove buffer and enzyme from the remainder of the restriction reactions by phenol extraction ethanol precipitation or using a similar DNA purification kit Resuspend the DNA in sterile dH2O Store the digested DNA at 20 C II Transfection Seed 2 x 10 cells in a 60 mm culture dish without antibiotics one day before transfection After 16 to 24 hours start transfection when the culture becomes 70 80 confluence Note We suggest transfecting cells with FuGENE Transfection Reagent Roche Applied Science or Lipofectamine Plus Invitrogen For example 4 ug of pacAd5 shuttle vector and I ug of pacAd5 9 2 100 Ad backbone vector are mixed with 9 uL FuGENE Transfection Reagent according to the manufacturer s recommendation The mixed DNA FuGENE complex is added by dropwise into the culture media Aspirate the media containing transfection reagent the next day and add 4 mL of complete culture medium After incubating for 7 days check for the presence of plaques If plate is ready for harvest gt 50 of cells lifted then collect the Crude Viral Lysate If not feed the cells with 1 mL of complete culture medium c
17. igh level of expression in mammalian cells e GFP Puro Fusion Marker to monitor cells positive for expression and stable selection with either GFP or puromycin resistance Related Products 1 AD 100 293AD Cell Line 2 AD 200 ViraDuctin Adenovirus Transduction Reagent 3 VPK 099 ViraBind Adenovirus Miniprep Kit 4 VPK 109 QuickTiter Adenovirus Titer Immunoassay Kit 5 VPK 110 QuickTiter Adenovirus Titer ELISA Kit 6 VPK 111 Rapid RCA Assay Kit 7 VPK 250 RAPAd Universal Adenoviral Expression System 8 VPK 251 RAPAd RSV Adenoviral Expression System 9 VPK 254 RAPAd CMV Adenoviral Bicistronic Expression System GFP Kit Components 1 pacAd5 miR GFP Puro Shuttle Vector Part No 325301 One 40 uL vial at 0 25 mg mL 2 pacAd5 9 2 100 Vector Part No 325002 One 40 uL vial at 0 25 mg mL 3 pacAdS CMV GFP Control Vector Part No 325004 One 40 uL vial at 0 25 mg mL 4 pacAd5 CMV ntLacZ Control Vector Part No 325202 One 40 uL vial at 0 25 mg mL CELL BIOLABS INC ae Materials Not Supplied 1 293 cells we recommend 293AD Cell Line Cat AD 100 for high titer production of recombinant adenovirus 2 293 Cell Culture Medium 3 Transfection Reagents 4 Pacl New England Biolabs Cat RO547L Storage Upon receipt store all kit components at 20 C Safety Considerations Remember that you will be working with samples containing infectious virus Follow the recommended NIH gui
18. ontinue to incubate at 37 C with CO On day 10 check for the presence of plaques If plate is ready for harvest gt 50 of cells lifted then collect the Crude Viral Lysate If not feed the cells with 1 mL of complete culture medium continue to incubate at 37 C with CO2 Keep checking plate for the presence of plaques Do not keep plate more than 15 days CELL BIOLABS INC a oa III Harvesting the Crude Viral Lysate 1 Harvest adenovirus containing cells by squirting cells off the plate with a 5 or 10 mL sterile serological pipette Transfer cells and media to a sterile 15 ml tube Scrape the cells into the medium with a cell lifter if necessary 2 Release viruses from cells by three freeze thaw cycles 10 minutes each in 37 C water bath and dry ice methanol bath 3 Centrifuge the cell lysate in a table top centrifuge at 3000 rpm for 15 minutes at room temperature to pellet the cell debris 4 Aliquot and store the Crude Viral Lysate Initial Viral Stock at 80 C IV Amplification Note The following procedure is suggested for T75 flasks and may be optimized to suit individual needs 1 Seed 3 5 x 10 cells in a T75 flask one day before infection 2 Add 50 of the above Crude Viral Lysate to the culture We recommend using a multiplicity of gt 0 5 PFU plaque forming units or enough viruses that cells demonstrate cytopathic effects CPEs within 48 hrs 3 During 24 48 hr infection examine the monolayer twic
19. port factor exportin 5 which transports them to the cytoplasm There the RNase II enzyme Dicer performs a second cleavage to generate a double stranded 18 24 nucleotide RNA molecule The RISC then associates with this RNA duplex and unwinds it Generally only one strand is stably incorporated into the RISC the other is discarded and rapidly degraded miRNAs guide the RISC to target messages that are subsequently cleaved or translationally silenced Figure 1 Gppp N AAAAA Drosha pri miRNA oO P P bt hes Gppp target cleavage translation repression Figure 1 miRNA Biogenesis and function Recombinant adenoviruses have tremendous potential in both research and therapeutic applications There are numerous advantages they provide when introducing genetic material into host cells The permissive host cell range is very wide The virus has been used to infect many mammalian cell types both replicative and non replicative for high expression of the recombinant protein Recombinant adenoviruses are especially useful for gene transfer and protein expression in cell lines that have low transfection efficiency with liposome After entering cells the virus remains epichromosomal i e does not integrate into the host chromosome so does not activate or inactivate host genes Recently recombinant adenoviruses have been used to deliver RNAi into cells 2 eo CELL BIOLABS INC Fe Cell Biolabs RA
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