pCDH1-MCS2-EF1-copGFP

pCDH1-MCS2-EF1-copGFP

pCDH1-MCS2-EF1-copGFP载体基本信息：

pCDH1-MCS2-EF1-copGFP载体质粒图谱和多克隆位点信息：

pCDH1-MCS2-EF1-copGFP载体简介：

背景简介：

This manual provides details and information necessary to generate expression constructs of your gene of interest in the pCDH cDNA Cloning and Expression

Lentivectors. Specifically, it provides critical instructions on amplification and cloning cDNA into the pCDH vectors, and verification of the final expression constructs. This manual does not include information on packaging the pCDH expression constructs into pseudotyped viral particles or transducing your target cells of choice with these particles. This information is available in the user manual Lentivector Expression Systems: Guide to Packaging and Transduction of Target Cells which is available on the SBI website. Before using the reagents and material supplied with this system, please read the entire manual.

基于HIV-1的pCDH 慢病毒载体特征：

 Multiple Cloning Site (MCS)—for cloning the gene of interest in the MCS located downstream of the CMV promoter.

 WPRE element—enhances stability and translation of the CMV-driven transcripts.

 SV40 polyadenylation signal—enables efficient termination of transcription and processing of recombinant transcripts.

 Hybrid RSV/5LTR promoter—provides a high level of expression of the full-length viral transcript in producer 293 cells.

 Genetic elements (cPPT, gag, env, LTRs)—necessary for packaging, transducing, and stably integrating the vira expression construct into genomic DNA.

 SV40 origin—for stable propagation of the pCDH plasmid in mammalian cells.

 pUC origin—for high copy replication and maintenance of the plasmid in E.coli cells.

 Ampicillin resistance gene—for selection in E.coli cells.

pCDH 慢病毒表达载体的优势：

Lentiviral expression vectors are the most effective vehicles for the delivery and expression of a gene of interest to almost any mammalian cell—including non-dividing cells and model organisms (C.A. Machida, 2003; M. Federico, 2003; W. C. Heiser, 2004). As with standard plasmid vectors, it is possible to introduce lentivector expression constructs in plasmid form into the cells with low-to-medium efficiency using conventional transfection protocols. However, by packaging the lentivector construct into viral particles, you can obtain highly efficient transduction of expression constructs—even with the most difficult to transfect cells, such as primary, stem, and differentiated cells. The expression construct transduced in target cells is integrated into genomic DNA and provides stable, long-term expression of the target gene.

pCDH 慢病毒载体的包装载体及细胞系

The expression lentivector contains the genetic elements responsible for packaging, transduction, stable integration of the viral expression construct into genomic DNA, and expression of the target gene sequence. The packaging vector provides all the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant viral particles. To produce a high titer of viral particles, expression and packaging vectors are transiently co-transfected into producer mammalian cells (e.g., HEK 293 cells). For a detailed description of SBI’s Lentivector expression system,please refer to the Lentivector Expression System user manual.

启动子的选择：

SBI provides a collection of cDNA cloning and expression vectors for various applications. A gene of interest can be cloned under a CMV or EF1 promoter with or without another expression cassette for a reporter gene (copGFP or PuroR). Genes can be either expressed transiently through transfection or stably expressed in a target cell line through transduction with packaged viral particles.

The major concern of cDNA expression in lentivectors is the efficiency level and stability of expression in target cell lines.

The Cytomegalovirus (CMV) promoter is a strong and most commonly used viral promoter that constitutively expresses downstream genes. While the CMV promoter works perfectly in the most common cell lines, it shows poor expression in some stem cell lines and hematopoietic cell lines (R.F. Doll, 1996; E.D. Papadakis, 2004).The housekeeping elongation factor 1α (EF1) promoter has been shown to exceed and outlast CMV-mediated expression in retroviral, lentiviral, and adenoviral vectors, in hematopoietic cell lines (K. Tokushige 1997; H. Nakai, 1998; C. Teschendorf, 2002). EF1 also performs well in most common cell lines.

MSCV promoter is the 5’-LTR promoter of murine stem cell virus. When a portion of the U3 region of the 3’ HIV LTR was replaced with the U3 region of MSCV LTR, the resulted hybrid HIV/MSCV LTR has dramatically increased the transgene expression level in human CD34+ hematopoietic cells (J.K. Choi, 2001). After integration into genomic DNA, this promoter transcribes a long transcript with an intron in the 5’UTR flanked with splice donor and acceptor sites derived from the lentiviral vector. Further studies found that additional CpG mutations in the MSCV LTR reduced transcriptional silencing in embryonic stem cells (C.S. Swindle, 2004). We constructed cDNA expression vectors with the CpG-deficient MSCV incorporated into the 3’ HIV LTR. After integration into genomic DNA, 3’MSCV/LTR will replace the 5’LTR and provide a high level of expression of the target gene and reporter gene downstream.

SBI第三代慢病毒载体

SBI offers a third generation of the most popular HIV-1 based lentivector expression system which consists of three maincomponents:

(1) The lentiviral expression vector (e.g., pCDH-EF1-MCS-T2A-Puro)

(2) The lentiviral packaging plasmids (e.g., pPACKH1 Packaging Plasmid mix)

(3) A pseudoviral particle producer cell line (e.g., 293TN cells)

2A Peptide-enabled dual expression system

Coexpression of a reporter gene together with a gene of interest is a useful approach for selecting transfected or transduced cells. This is commonly achieved by using two independent internal promoters, such as CMV and EF1 in pCDH-CMV-MCSEF1- copGFP, or by linking two transgenes with an internal ribosomal entry site (IRES) element in a single bicistronic transcript. Many dual promoter pairs have shown a high level of expression of both transgenes in standard cell lines— however, promoter interference often occurs in some cell lines. There are also two main problems that limit the use of IRES: the large size and the imbalanced expression between the first and second cistrons (H. Mizuguchi, 2000; X.Yu, 2003).

The “self-cleaving” 2A peptides have been used successfully to generate multiple proteins from a single promoter in many applications (P. de Felipe, 2004; M.J. Osborn, 2005; P. de Felipe, 2006). The 2A-like sequences exist in several  viruses  and are used to mediate protein cleavage from a single open reading frame. Through a ribosomal skip mechanism, the 2A peptide prevents normal peptide bond formation between the 2A glycine and the 2B proline without affecting the translation of 2B (M.L. Donnelly, 2001):SBI’s cDNA expression vectors incorporate the 2A-like sequence (T2A) from the insect virus Thosea asigna to mediate the coexpression of a reporter gene with the target cDNA. Reporter genes have been cloned at either the first or second positions, and we achieved high expression levels at both locations.

pCDH1-MCS2-EF1-copGFP载体序列

ORIGIN

    1 CATATGCCAA GTACGCCCCC TATTGACGTC AATGACGGTA AATGGCCCGC CTGGCATTAT

   61 GCCCAGTACA TGACCTTATG GGACTTTCCT ACTTGGCAGT ACATCTACGT ATTAGTCATC

  121 GCTATTACCA TGGTGATGCG GTTTTGGCAG TACATCAATG GGCGTGGATA GCGGTTTGAC

  181 TCACGGGGAT TTCCAAGTCT CCACCCCATT GACGTCAATG GGAGTTTGTT TTGGCACCAA

  241 AATCAACGGG ACTTTCCAAA ATGTCGTAAC AACTCCGCCC CATTGACGCA AATGGGCGGT

  301 AGGCGTGTAC GGTGGGAGGT CTATATAAGC AGAGCTTGTG AAACTTCGAG GAGTCTCTTT

  361 GTTGAGGACT TTTGAGTTCT CCCTTGAGGC TCCCACAGAT ACAATAAATA TTTGAGATTG

  421 AACCCTGTCG AGTATCTGTG TAATCTTTTT TACCTGTGAG GTCTCGGAAT CCGGGCCGAG

  481 AACTTCGCAG TTGGCGCCCG AACAGGGACT TGATTGAGAG TGATTGAGGA AGTGAAGCTA

  541 GAGCAATAGA AAGCTGTTAA GCAGAACTCC TGCTGACCTA AATAGGGAAG CAGTAGCAGA

  601 CGCTGCTAAC AGTGAGTATC TCTAGTGAAG CAGACTCGAG CTCATAATCA AGTCATTGTT

  661 TAAAGGCCCA GATAAATTAC ATCTGGTGAC TCTTCGCGGA CCTTCAAGCC AGGAGATTCG

  721 CCGAGGGACA GTCAACAAGG TAGGAGAGAT TCTACAGCAA CATGGGGAAT GGACAGGGGC

  781 GAGATTGGAA AATGGCCATT AAGAGATGTA GTAATGTTGC TGTAGGAGTA GGGGGGAAGA

  841 GTAAAAAATT TGGAGAAGGG AATTTCAGAT GGGCCATTAG AATGGCTAAT GTATCTACAG

  901 GACGAGAACC TGGTGATATA CCAGAGACTT TAGATCAACT AAGGTTGGTT ATTTGCGATT

  961 TACAAGAAAG AAGAGAAAAA TTTGGATCTA GCAAAGAAAT TGATATGGCA ATTCCTGCAT

 1021 TGAGGAGAAA TGGTAGGCAA TGTGGCATGT CTGAAAAAGA GGAGGAATGA TGAAGTATCT

 1081 CAGACTTATT TTATAAGGGA GATACTGTGC TGAGTTCTTC CCTTTGAGGA AGGTATGTCA

 1141 TATCCTAGAC ATAGTCTCAA TTTTAAAAGA AGAGGTAGGA TAGGAGGGAT GGCCCCTTAT

 1201 GAATTATTAG CACAACAAGA ATCCTTAAGA ATACAAGATT ATTTTTCTGC AATACCACAA

 1261 AAATTGCAAG CACAGTGGAT TTATTATAAA GATCAAAAAG ATAAGAAATG GAAAGGACCA

 1321 ATGAGAGTAG AATACTGGGG ACAGGGATCA GTATTATTAA AGGATGAAGA GAAGGGATAT

 1381 TTTCTTATAA TCGATACTAG TATTATGCCC AGTACATGAC CTTATGGGAC TTTCCTACTT

 1441 GGCAGTACAT CTACGTATTA GTCATCGCTA TTACCATGGT GATGCGGTTT TGGCAGTACA

 1501 TCAATGGGCG TGGATAGCGG TTTGACTCAC GGGGATTTCC AAGTCTCCAC CCCATTGACG

 1561 TCAATGGGAG TTTGTTTTGG CACCAAAATC AACGGGACTT TCCAAAATGT CGTAACAACT

 1621 CCGCCCCATT GACGCAAATG GGCGGTAGGC GTGTACGGTG GGAGGTCTAT ATAAGCAGAG

 1681 CTCGTTTAGT GAACCGTCAG ATCGCCTGGA GACGCCATCC ACGCTGTTTT GACCTCCATA

 1741 GAAGATTCTA GAGCCCGGGC GCGCCGGATC CAGATCTTAA TTAATTTAAA TGAATTCGCG

 1801 GCCGCGAAGG ATCTGCGATC GCTCCGGTGC CCGTCAGTGG GCAGAGCGCA CATCGCCCAC

 1861 AGTCCCCGAG AAGTTGGGGG GAGGGGTCGG CAATTGAACG GGTGCCTAGA GAAGGTGGCG

 1921 CGGGGTAAAC TGGGAAAGTG ATGTCGTGTA CTGGCTCCGC CTTTTTCCCG AGGGTGGGGG

 1981 AGAACCGTAT ATAAGTGCAG TAGTCGCCGT GAACGTTCTT TTTCGCAACG GGTTTGCCGC

 2041 CAGAACACAG CTGAAGCTTC GAGGGGCTCG CATCTCTCCT TCACGCGCCC GCCGCCCTAC

 2101 CTGAGGCCGC CATCCACGCC GGTTGAGTCG CGTTCTGCCG CCTCCCGCCT GTGGTGCCTC

 2161 CTGAACTGCG TCCGCCGTCT AGGTAAGTTT AAAGCTCAGG TCGAGACCGG GCCTTTGTCC

 2221 GGCGCTCCCT TGGAGCCTAC CTAGACTCAG CCGGCTCTCC ACGCTTTGCC TGACCCTGCT

 2281 TGCTCAACTC TACGTCTTTG TTTCGTTTTC TGTTCTGCGC CGTTACAGAT CCAAGCTGTG

 2341 ACCGGCGCCT ACGCTAGACG CCACCATGGA GAGCGACGAG AGCGGCCTGC CCGCCATGGA

 2401 GATCGAGTGC CGCATCACCG GCACCCTGAA CGGCGTGGAG TTCGAGCTGG TGGGCGGCGG

 2461 AGAGGGCACC CCCAAGCAGG GCCGCATGAC CAACAAGATG AAGAGCACCA AAGGCGCCCT

 2521 GACCTTCAGC CCCTACCTGC TGAGCCACGT GATGGGCTAC GGCTTCTACC ACTTCGGCAC

 2581 CTACCCCAGC GGCTACGAGA ACCCCTTCCT GCACGCCATC AACAACGGCG GCTACACCAA

 2641 CACCCGCATC GAGAAGTACG AGGACGGCGG CGTGCTGCAC GTGAGCTTCA GCTACCGCTA

 2701 CGAGGCCGGC CGCGTGATCG GCGACTTCAA GGTGGTGGGC ACCGGCTTCC CCGAGGACAG

 2761 CGTGATCTTC ACCGACAAGA TCATCCGCAG CAACGCCACC GTGGAGCACC TGCACCCCAT

 2821 GGGCGATAAC GTGCTGGTGG GCAGCTTCGC CCGCACCTTC AGCCTGCGCG ACGGCGGCTA

 2881 CTACAGCTTC GTGGTGGACA GCCACATGCA CTTCAAGAGC GCCATCCACC CCAGCATCCT

 2941 GCAGAACGGG GGCCCCATGT TCGCCTTCCG CCGCGTGGAG GAGCTGCACA GCAACACCGA

 3001 GCTGGGCATC GTGGAGTACC AGCACGCCTT CAAGACCCCC ATCGCCTTCG CCAGATCCCG

 3061 CGCTCAGTCG TCCAATTCTG CCGTGGACGG CACCGCCGGA CCCGGCTCCA CCGGATCTCG

 3121 CTAAGTCGAC AATCAACCTC TGGATTACAA AATTTGTGAA AGATTGACTG GTATTCTTAA

 3181 CTATGTTGCT CCTTTTACGC TATGTGGATA CGCTGCTTTA ATGCCTTTGT ATCATGCTAT

 3241 TGCTTCCCGT ATGGCTTTCA TTTTCTCCTC CTTGTATAAA TCCTGGTTGC TGTCTCTTTA

 3301 TGAGGAGTTG TGGCCCGTTG TCAGGCAACG TGGCGTGGTG TGCACTGTGT TTGCTGACGC

 3361 AACCCCCACT GGTTGGGGCA TTGCCACCAC CTGTCAGCTC CTTTCCGGGA CTTTCGCTTT

 3421 CCCCCTCCCT ATTGCCACGG CGGAACTCAT CGCCGCCTGC CTTGCCCGCT GCTGGACAGG

 3481 GGCTCGGCTG TTGGGCACTG ACAATTCCGT GGTGTTGTCG GGGAAATCAT CGTCCTTTCC

 3541 TTGGCTGCTC GCCTGTGTTG CCACCTGGAT TCTGCGCGGG ACGTCCTTCT GCTACGTCCC

 3601 TTCGGCCCTC AATCCAGCGG ACCTTCCTTC CCGCGGCCTG CTGCCGGCTC TGCGGCCTCT

 3661 TCCGCGTCTT CGCCTTCGCC CTCAGACGAG TCGGATCTCC CTTTGGGCCG CCTCCCCGCC

 3721 GGTACCGATG ACAGAGTTAG AAGATCGCTT CAGGAAGCTA TTTGGCACGA CTTCTACAAC

 3781 GGGAGACAGC ACAGTAGATT CTGAAGATGA ACCTCCTAAA AAAGAAAAAA GGGTGGACTG

 3841 GGATGAGTAT TGGAACCCTG AAATCGATAG CTTCCAGTGC TTTGTGAAAC TTCGAGGAGT

 3901 CTCTTTGTTG AGGACTTTTG AGTTCTCCCT TGAGGCTCCC ACAGATACAA TAAATATTTG

 3961 AGATTGAACC CTGTCGAGTA TCTGTGTAAT CTTTTTTACC TGTGAGGTCT CGGAATCCGG

 4021 GCCGAGAACT TCGCAGCGAG CTCATTGTAC CGCGAACTTG TTTATTGCAG CTTATAATGG

 4081 TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCATTTTTTT CACTGCATTC

 4141 TAGTTGTGGT TTGTCCAAAC TCATCAATGT ATCTTATCAT GTCTGGCTCT AGCTATCCCG

 4201 CCCCTAACTC CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT

 4261 TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG AGGAGGCTTT

 4321 TTTGGAGGCC TAGACTTTTG CAGAGACGGC CCAAATTCGT AATCATGGTC ATAGCTGTTT

 4381 CCTGTGTGAA ATTGTTATCC GCTCACAATT CCACACAACA TACGAGCCGG AAGCATAAAG

 4441 TGTAAAGCCT GGGGTGCCTA ATGAGTGAGC TAACTCACAT TAATTGCGTT GCGCTCACTG

 4501 CCCGCTTTCC AGTCGGGAAA CCTGTCGTGC CAGCTGCATT AATGAATCGG CCAACGCGCG

 4561 GGGAGAGGCG GTTTGCGTAT TGGGCGCTCT TCCGCTTCCT CGCTCACTGA CTCGCTGCGC

 4621 TCGGTCGTTC GGCTGCGGCG AGCGGTATCA GCTCACTCAA AGGCGGTAAT ACGGTTATCC

 4681 ACAGAATCAG GGGATAACGC AGGAAAGAAC ATGTGAGCAA AAGGCCAGCA AAAGGCCAGG

 4741 AACCGTAAAA AGGCCGCGTT GCTGGCGTTT TTCCATAGGC TCCGCCCCCC TGACGAGCAT

 4801 CACAAAAATC GACGCTCAAG TCAGAGGTGG CGAAACCCGA CAGGACTATA AAGATACCAG

 4861 GCGTTTCCCC CTGGAAGCTC CCTCGTGCGC TCTCCTGTTC CGACCCTGCC GCTTACCGGA

 4921 TACCTGTCCG CCTTTCTCCC TTCGGGAAGC GTGGCGCTTT CTCATAGCTC ACGCTGTAGG

 4981 TATCTCAGTT CGGTGTAGGT CGTTCGCTCC AAGCTGGGCT GTGTGCACGA ACCCCCCGTT

 5041 CAGCCCGACC GCTGCGCCTT ATCCGGTAAC TATCGTCTTG AGTCCAACCC GGTAAGACAC

 5101 GACTTATCGC CACTGGCAGC AGCCACTGGT AACAGGATTA GCAGAGCGAG GTATGTAGGC

 5161 GGTGCTACAG AGTTCTTGAA GTGGTGGCCT AACTACGGCT ACACTAGAAG GACAGTATTT

 5221 GGTATCTGCG CTCTGCTGAA GCCAGTTACC TTCGGAAAAA GAGTTGGTAG CTCTTGATCC

 5281 GGCAAACAAA CCACCGCTGG TAGCGGTGGT TTTTTTGTTT GCAAGCAGCA GATTACGCGC

 5341 AGAAAAAAAG GATCTCAAGA AGATCCTTTG ATCTTTTCTA CGGGGTCTGA CGCTCAGTGG

 5401 AACGAAAACT CACGTTAAGG GATTTTGGTC ATGAGATTAT CAAAAAGGAT CTTCACCTAG

 5461 ATCCTTTTAA ATTAAAAATG AAGTTTTAAA TCAATCTAAA GTATATATGA GTAAACTTGG

 5521 TCTGACAGTT ACCAATGCTT AATCAGTGAG GCACCTATCT CAGCGATCTG TCTATTTCGT

 5581 TCATCCATAG TTGCCTGACT CCCCGTCGTG TAGATAACTA CGATACGGGA GGGCTTACCA

 5641 TCTGGCCCCA GTGCTGCAAT GATACCGCGA GACCCACGCT CACCGGCTCC AGATTTATCA

 5701 GCAATAAACC AGCCAGCCGG AAGGGCCGAG CGCAGAAGTG GTCCTGCAAC TTTATCCGCC

 5761 TCCATCCAGT CTATTAATTG TTGCCGGGAA GCTAGAGTAA GTAGTTCGCC AGTTAATAGT

 5821 TTGCGCAACG TTGTTGCCAT TGCTACAGGC ATCGTGGTGT CACGCTCGTC GTTTGGTATG

 5881 GCTTCATTCA GCTCCGGTTC CCAACGATCA AGGCGAGTTA CATGATCCCC CATGTTGTGC

 5941 AAAAAAGCGG TTAGCTCCTT CGGTCCTCCG ATCGTTGTCA GAAGTAAGTT GGCCGCAGTG

 6001 TTATCACTCA TGGTTATGGC AGCACTGCAT AATTCTCTTA CTGTCATGCC ATCCGTAAGA

 6061 TGCTTTTCTG TGACTGGTGA GTACTCAACC AAGTCATTCT GAGAATAGTG TATGCGGCGA

 6121 CCGAGTTGCT CTTGCCCGGC GTCAATACGG GATAATACCG CGCCACATAG CAGAACTTTA

 6181 AAAGTGCTCA TCATTGGAAA ACGTTCTTCG GGGCGAAAAC TCTCAAGGAT CTTACCGCTG

 6241 TTGAGATCCA GTTCGATGTA ACCCACTCGT GCACCCAACT GATCTTCAGC ATCTTTTACT

 6301 TTCACCAGCG TTTCTGGGTG AGCAAAAACA GGAAGGCAAA ATGCCGCAAA AAAGGGAATA

 6361 AGGGCGACAC GGAAATGTTG AATACTCATA CTCTTCCTTT TTCAATATTA TTGAAGCATT

 6421 TATCAGGGTT ATTGTCTCAT GAGCGGATAC ATATTTGAAT GTATTTAGAA AAATAAACAA

 6481 ATAGGGGTTC CGCGCACATT TCCCCGAAAA GTGCCACCTG ACGTCTAAGA AACCATTATT

 6541 ATCATGACAT TAACCTATAA AAATAGGCGT ATCACGAGGC CCTTTCGTCT CGCGCGTTTC

 6601 GGTGATGACG GTGAAAACCT CTGACACATG CAGCTCCCGG AGACGGTCAC AGCTTGTCTG

 6661 TAAGCGGATG CCGGGAGCAG ACAAGCCCGT CAGGGCGCGT CAGCGGGTGT TGGCGGGTGT

 6721 CGGGGCTGGC TTAACTATGC GGCATCAGAG CAGATTGTAC TGAGAGTGCA C//

pCDH1-MCS2-EF1-copGFP其他相关慢病毒载体：