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Blood, Vol. 111, Issue 4, 1866-1875, February 15, 2008
An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation
Blood Ryu et al.
111: 1866
Supplemental materials for: Ryu et al
Plasmid Construction: A plasmid for a self-complementary (sc) AAV-GFP vector (pscAAV-GFP) was constructed from pscAAV-LP1-hFIXco1 by replacing the 452 bp Tsp45 I – BspE I fragment containing the LP1 promoter with a 382 bp PCR product containing the CMV promoter and by replacing the hFIXco coding sequence with an EcoRI – NotI fragment containing the enhanced, humanized GFP coding sequences (derived from pEGFP-N1, Clonetech). An AAV vector encoding Cre-recombinase (pscAAV-Cre) was constructed from pscAAV-GFP by replacing the EcoRI – NotI GFP fragment with a PCR amplified Cre recombinase coding sequence derived from pMC-CreN kindly provided by Dr. Fred Alt (Children’s Hospital, Boston). For the initial cassette exchange, pLTR-REDflox was constructed by replacing the GFP coding sequence of pLTR-GFPflox with those of DsRed2 (Clonetech). To construct exchange cassettes having one copy of the insulator, either the 1.2kb cHS4 or the 2 × 250bp core chromatin insulator fragment was inserted into the unique BglII site of pLTR-GFPflox to place the insulator fragment between the LTR and the loxP site. These insulator elements were also inserted into a unique SalI site between the loxP511 and the polyA signal in pLTR-GFPflox to make the 5′ insulator constructs. Cassettes in which the insulator elements flanked the LTR-GFP transgene were then generated by combining the 5′ and 3′ insulator constructs in a single plasmid using unique restriction enzyme sites that were shared between the two plasmids. The globin LCR cassette was constructed in two steps. First the  -globin coding sequences in the plasmid pCL20cmLAR  V52 were replaced with GFP coding sequences and then a fragment that included the globin LCR , the -globin promoter and GFP coding sequences were used to replace LTR-GFP in the pLTR-GFPflox plasmid. To construct the proviral cassette with 2 LTRs, a 3.8kb PciI/ScaI fragment from MFGS-B2-IL2RG, a vector made in the laboratory of Brian Sorrentino3 using the MFG backbone4, was blunt-ended and ligated to the blunt-ended 2.7kb BglII/SalI fragment from pLTR-GFP. Then, the IL2Rc coding sequences and 3′LTR were replaced with GFP-3′MFG LTR sequences to make a floxed proviral GFP expression cassette. For the lentiviral cassette having the internal EF1 promoter driving GFP expression with insulators in the SIN LTRs, a fragment containing either the 5′ or 3′ SIN LTR with insulator (cHS4) was PCR-amplified from a transduced Jurkat clone5, cloned into TOPO-TA vector, and the sequence of each was verified. The 5′ fragment was used to replace the MFG LTR in pLTR-GFP (p5INS-flox). The 3′ SIN LTR with insulator was used to replace the 3′ SIN LTR from the pCL20c EF1a-GFP vector6, after which the BssHII/XhoI fragment was ligated to the BssHII/SalI fragment of p5INS-flox. Analysis of Gene Expression: Total RNA was extracted using RNA-Bee (Tel-Test, Friendswood, TX). VIC-labeled GAPDH (Applied Biosystems), and FAM-labeled LMO2 (Applied Biosystems) were added to the TaqMan One-Step real-time PCR Master mix which was processed on an ABI 7900HT system using the following PCR conditions: an initial reverse transcription at 25°C for 10 min and 48°C for 30 min, and then at 95°C for 10 min followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The comparative threshold cycle (CT) method was used with the values normalized to GAPDH and compared to results with positive control K562 cells. For Northern blot analysis, 10 µg total RNA samples from clones or K562 cells were separated in a 1% formaldehyde gel. Samples were then transferred onto a GeneScreen Plus membrane (Perkin-Elmer), and hybridized with 480bp LMO2 fragment prepared from a human LMO2 cDNA clone pCMV6-XL5 (OriGene Technologies, Rockville, MD). Forty micrograms of protein were separated on a 10% NuPage gel (Invitrogen) and transferred onto a nitrocellulose PVDF membrane (Invitrogen). The membrane was cut between 37 and 50 kD and blocked in milk. The top part of the membrane was immunoblotted with HRP-conjugated antibody against -actin (Santa Cruz Biotech, Santa Cruz, CA) while the bottom part was incubated with anti-human LMO2 antibody (R&D Systems, Minneapolis, MN) followed by HRP-conjugated secondary antibody (Calbiochem, San Diego, CA). Signals were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Pierce). REFERENCES 1. Nathwani AC, Gray JT, Ng CY, et al. Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood. 2006;107:2653-2661.
2. Hanawa H, Hargrove PW, Kepes S, Srivastava DK, Nienhuis AW, Persons DA. Extended beta-globin locus control region elements promote consistent therapeutic expression of a gamma-globin lentiviral vector in murine beta-thalassemia. Blood. 2004;104:2281-2290.
3. Shou Y, Ma Z, Lu T, Sorrentino BP. Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy. Proc Natl Acad Sci USA. 2006;103:11730-5.
4. Riviere I, Brose K, Mulligan RC. Effects of retroviral vector design on expression of human adenosine deaminase in murine bone marrow transplant recipients engrafted with genetically modified cells. Proc Natl Acad Sci USA. 1995;92:6733-7.
5. Evans-Galea MV, Wielgosz MM, Hanawa H, Srivastava DK, Nienhuis AW. Suppression of Clonal Dominance in Cultured Human Lymphoid Cells by Addition of the cHS4 Insulator to a Lentiviral Vector. Mol Ther. 2007;15:801-9.
6. Hanawa H, Persons DA, Nienhuis AW. Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors. J Virol. 2005;79:8410-8421.
Files in this Data Supplement:
- Table S1. Primer Sequences (JPG, 88.3 KB)
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- Table S2. Efficiency of targeting and subsequent cassette exchange (JPG, 108 KB)
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- Figure S1. Southern blot analysis of selected clones after cassette exchange with flanking 1.2kb insulator construct (JPG, 86.6 KB)
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(A) The expected band sizes are illustrated. D: DraIII and K: KpnI. The probes are indicated as double lines under the diagram. (B) Genomic DNA digested with DraIII (left) or KpnI (right) and probed with the LMO2 probe (upper panel) or the GFP probe (lower panel). The red arrows mark the predicted band for exchanged allele whereas blue arrows indicate the unmodified wild-type allele.
- Figure S2. Southern blot analysis of selected clones after cassette exchange with flanking 2 × 250bp core insulator construct (JPG, 93.5 KB)
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(A) The expected band sizes are illustrated. D: DraIII and B: BglII. The probes are indicated as double lines under the diagram. (B) Either DraIII (left) or BglII (right) blot was probed with the LMO2 probe (upper panel) or the GFP probe (lower panel). The red arrows mark the predicted band for exchanged allele whereas blue arrows indicate the unmodified wild-type allele.
- Figure S3. Southern blot analysis of selected clones after cassette exchange with 5′ single copy insulator construct (JPG, 94.2 KB)
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(A) The expected band sizes are illustrated. D: DraIII and H: HindIII. The probes are indicated as double lines under the diagram. (B) Either DraIII (left) or HindIII (right) blot was probed with the LMO2 probe (upper panel) or the GFP probe (lower panel). The red arrows mark the predicted band for exchanged allele whereas blue arrows indicate the unmodified wild-type allele.
- Figure S4. Southern blot analysis of selected clones after cassette exchange with 3′ single copy insulator construct (JPG, 109 KB)
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(A) The expected band sizes are illustrated. D: DraIII and H: HindIII. The probes are indicated as double lines under the diagram. (B) Either DraIII (left) or HindIII (right) blot was probed with the LMO2 probe (upper panel) or the GFP probe (lower panel). The red arrows mark the predicted band for exchanged allele whereas blue arrows indicate the unmodified wild-type allele.
- Figure S5. The effect of single copy insulator on LMO2 activation and GFP expression (JPG, 96 KB)
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(A) qRT-PCR on RNA samples from selected clones. When insulator was inserted between LTR and proximal promoter (3′ single copy; green bar), LMO2 activation was reduced compared to uninsulated original targeted clones. Mean ± standard deviation of clones are indicated above the bar graph. (B) Flow analysis of selected clones. 3′ single copy insulator clones also showed reduced GFP expression. Average MFI of clones are shown. P values were calculated using two-tailed t-test.
- Figure S6. Southern blot analysis of selected clones after cassette exchange with globin LCR construct (JPG, 78.1 KB)
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(A) The expected band sizes are illustrated. B: BgiII and H: HindIII. The probes are indicated as double lines under the diagram. (B) Either HindIII (left) or BglII (right) blot was probed with the LMO2 probe (upper panel) or the GFP probe (lower panel). The red arrows mark the predicted band for exchanged allele whereas blue arrows indicate the unmodified wild-type allele.
- Figure S7. Southern blot analysis of selected clones after cassette exchange with proviral construct (JPG, 88.8 KB)
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(A) The expected band sizes are illustrated. D: DraIII and N: NcoI. The probes are indicated as double lines under the diagram. (B) Genomic DNA digested with DraIII (left) or NcoI (right) and probed with the LMO2 probe (upper panel) or the GFP probe (lower panel). The red arrows mark the predicted band for exchanged allele whereas blue arrows indicate the unmodified wild-type allele.
- Figure S8. Southern blot analysis of selected clones after cassette exchange with proviral construct (JPG, 91.3 KB)
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(A) The expected band sizes are illustrated. D: DraIII and N: NcoI. The probes are indicated as double lines under the diagram. (B) Genomic DNA digested with DraIII (left) or NcoI (right) and probed with the LMO2 probe (upper panel) or the GFP probe (lower panel). The red arrows mark the predicted band for exchanged allele whereas blue arrows indicate the unmodified wild-type allele.
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