- Email: [email protected]
1153. Strand Bias in Gene Repair Using Single-Stranded Oligonucleotides
GENE REGULATION: PROMOTER AND CONSTRUCT DESIGN egr-1 promoter. Material and Methods. Adenoviral vectors were generated based on the AdEasy system and homologous recombination in bacterials. –600 pb. fragment of egr-1 promoter was cloned upstream of the luciferase gene into the MCS of pShuttle plasmid. Adenoviral plasmids resulting was screened, sequenced and packaged into HEK293 cells using effectene transfection system (Quiagen). Adenoviral large scale stock was screened, purified and titer for further experiments. Glioma cell lines (CH235, D54 and U373) were grown in D-MEM media containing 10% of FBS and 1% Antibiotics in standard conditions. 1x 105 cells were seeded and infected with 25 MOI for 2 hours in low serum media. After that, cells were washed and maintained in D-MEM media supplemented with 1 % of FBS for 24 hrs before to start induction with 100 ng/ml of Betamethazone, 100 ng/ml of b-Estradiol and 50 ng/ml of Progesterone as previously reported. Unstimulated and infected cells were maintained in 1 % FBS and used as basal control. Proteins were purified using Cell Culture Lysis (Promega) and stored at –20° C. Luciferase Activity was quantified using Luciferase Assay System according to manufacturer instructions (Promega coorp.) in a Victor Wallac 2 instrument. All experiments were performed for triplicate. Results and conclusions. In CH235 cell line induction of activity reaches a maximum expression six hours after induction with BEstradiol and this level is maintained until 12 hrs. These activities compared to basal expression means 2.7 holds of luciferase activity. D54 cells not show significative differences of luciferase activity compared to basal with three inductors. Finally, U373 cells show a slightly increase of luciferase activity with Betamethazone, bEstradiol and Progesterone at 6 hours after induction.
1152. Characterization of Copy Numbers of βActin and IFNγγ Genes in Mouse Tissues with Real Time Quantitative PCR Baohong Tian,1 Sarah S. Rentz, Amanda P. Cunningham, Tina Rogers, John G. Page. 1 Safety Assessment Department, Southern Research Institute, Birmingham, AL, United States. Characterization of copy numbers of some specific DNA sequences in animal tissues appeared to be impractical until the presence of real time quantitative PCR (Q-PCR). Since both βActin and IFNγ are highly interesting genes with a substantially different amount of gene products, we designed Molecular Beacon probes and their corresponding primers for both sequences to characterize their copy numbers in mouse tissues. Tissues from both male and female mice, including cerebrum, heart, lung, kidney, liver, spleen, stomach, small intestine, ovary and testes, were collected and lysed through homogenization in GITC solution. DNA was isolated with QIAGEN DNeasy Tissue Kit (Valencia, CA) and quantitated with PicoGreen (Molecular Probes, Eugene, OR). The copy numbers of both standards, a commercial DECAtemplate (Ambion, Austin, TX) containing mouse β-Actin cDNA sequence and a plasmid containing mouse IFNγ sequence, were calculated on the basis of the size and concentration and validated by limiting dilution assay (LDA). The copy numbers of β-Actin and IFNγ in a variety of tissues, based on their standards, were detected with real time Q-PCR (LightCycler, Roche Molecular Biochemicals, Mannheim, Germany), and then normalized on the basis of the DNA amount. In addition, two DNA samples from cerebrum and ovary were tested with LDA to validate the copy numbers of both β-Actin and IFNγ. The copy numbers of the standards validated by LDA with QPCR were consistent with the calculated copy numbers. Based on the standard curve, the copy numbers of β-Actin and IFNγ detected in a variety of tissues from four mice and normalized with DNA amount were constant among different tissues for both β-Actin and IFNγ. Surprisingly, the copy numbers of mouse β-Actin detected in S444
all these tissues was over tenfold higher than the expected two copies per 6.6 pg DNA (DNA amount per cell). In contrast, the copy numbers of mouse IFNγ had an average of 3 copies per 6.6 pg DNA, close to the expected two copies. Furthermore, LDA for both β-Actin and IFNγ in cerebrum and ovary supported the result that the same amount of DNA had tenfold different copy numbers between β-Actin and IFNγ. The following table shows the copy numbers of both β-Actin and IFNγ per 6.6 pg DNA in ten tissues from four mice (Mean±SE). Tissues Cerebrum Heart Lung Kidney Liver
Copies / 6.6 pg DNA β-Actin IFNγ 38.4±3.0 3.1±0.8 39.0±2.7 3.1±0.8 39.6±4.3 3.5±0.7 36.6±2.7 3.3±0.8 34.2±3.4 3.2±0.7
Tissues Spleen Stomach Small Intestine Ovary Testes
Copies / 6.6 pg DNA β-Actin IFNγ 31.2±5.0 3.2±0.6 35.4±2.9 3.3±0.9 36.2±2.7 3.4±0.8 29.9±4.9 2.0±0.1 34.6±2.3 3.6±0.8
This result successfully demonstrates that multiple gene sets of mouse β-Actin related sequences, possibly including many pseudogenes, were detected in mouse genome with Q-PCR. Additionally, a single set of mouse IFNγ was also quantitated as predicted. Altogether, this study validates the sensitivity, specificity, and accuracy of Q-PCR technology and supports its utility for its extensive applications.
1153. Strand Bias in Gene Repair Using SingleStranded Oligonucleotides Charlotte B. Sorensen,1 Anne-Margrethe Krogsdam,2 Karsten Kristiansen,2 Lars Bolund,1 Thomas G. Jensen.1 1 Department of Human Genetics, University of Aarhus, Aarhus, Denmark; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. Targeted gene conversion is a method developed for site-specific correction of dysfunctional target genes. The method is based on the interaction of either chimeric RNA/DNA or modified single-stranded oligonucleotides with their homologous target genes thereby generating a single mismatched nucleotide base pair. This mismatch is believed to be recognized by the endogenous repair system resulting in excision and substitution of the mismatched base using the oligonucleotide sequence as template. Although the potential of this technique is enormous, it still faces many obstacles such as the relatively low correction efficiencies obtained as well as low reproducibility among and even within laboratories. In order to optimize the efficiency, we are investigating the effect of substituting deoxyribonucleotides in single-stranded oligonucleotides with synthetic DNA-analogues with increased affinity and specificity. As a model system for testing designs of oligonucleotides, we use an episomally expressed beta-galactosidase gene. Oligonuclotides of varying lengths and polarity have been investigated with regard to correction efficiency. Based on the most efficient design, new oligo designs with synthetic DNA-analogues have been made and are being tested. It has previously been shown that antisense oriented singlestranded oligonucleotides results in a higher conversion frequency compared to its sense oriented counterpart. Using standard DNAcontaining oligonucleotides, we have found that this holds true for only one of the beta-galactosidase mutations studied. For the other mutation, the highest efficiency is obtained using the sense oriented oligonucleotide suggesting that limited accessibility due to presence of transcription complexes is not the only cause of the observed strand bias.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy
1152. Characterization of Copy Numbers of βActin and IFNγγ Genes in Mouse Tissues with Real Time Quantitative PCR Baohong Tian,1 Sarah S. Rentz, Amanda P. Cunningham, Tina Rogers, John G. Page. 1 Safety Assessment Department, Southern Research Institute, Birmingham, AL, United States. Characterization of copy numbers of some specific DNA sequences in animal tissues appeared to be impractical until the presence of real time quantitative PCR (Q-PCR). Since both βActin and IFNγ are highly interesting genes with a substantially different amount of gene products, we designed Molecular Beacon probes and their corresponding primers for both sequences to characterize their copy numbers in mouse tissues. Tissues from both male and female mice, including cerebrum, heart, lung, kidney, liver, spleen, stomach, small intestine, ovary and testes, were collected and lysed through homogenization in GITC solution. DNA was isolated with QIAGEN DNeasy Tissue Kit (Valencia, CA) and quantitated with PicoGreen (Molecular Probes, Eugene, OR). The copy numbers of both standards, a commercial DECAtemplate (Ambion, Austin, TX) containing mouse β-Actin cDNA sequence and a plasmid containing mouse IFNγ sequence, were calculated on the basis of the size and concentration and validated by limiting dilution assay (LDA). The copy numbers of β-Actin and IFNγ in a variety of tissues, based on their standards, were detected with real time Q-PCR (LightCycler, Roche Molecular Biochemicals, Mannheim, Germany), and then normalized on the basis of the DNA amount. In addition, two DNA samples from cerebrum and ovary were tested with LDA to validate the copy numbers of both β-Actin and IFNγ. The copy numbers of the standards validated by LDA with QPCR were consistent with the calculated copy numbers. Based on the standard curve, the copy numbers of β-Actin and IFNγ detected in a variety of tissues from four mice and normalized with DNA amount were constant among different tissues for both β-Actin and IFNγ. Surprisingly, the copy numbers of mouse β-Actin detected in S444
all these tissues was over tenfold higher than the expected two copies per 6.6 pg DNA (DNA amount per cell). In contrast, the copy numbers of mouse IFNγ had an average of 3 copies per 6.6 pg DNA, close to the expected two copies. Furthermore, LDA for both β-Actin and IFNγ in cerebrum and ovary supported the result that the same amount of DNA had tenfold different copy numbers between β-Actin and IFNγ. The following table shows the copy numbers of both β-Actin and IFNγ per 6.6 pg DNA in ten tissues from four mice (Mean±SE). Tissues Cerebrum Heart Lung Kidney Liver
Copies / 6.6 pg DNA β-Actin IFNγ 38.4±3.0 3.1±0.8 39.0±2.7 3.1±0.8 39.6±4.3 3.5±0.7 36.6±2.7 3.3±0.8 34.2±3.4 3.2±0.7
Tissues Spleen Stomach Small Intestine Ovary Testes
Copies / 6.6 pg DNA β-Actin IFNγ 31.2±5.0 3.2±0.6 35.4±2.9 3.3±0.9 36.2±2.7 3.4±0.8 29.9±4.9 2.0±0.1 34.6±2.3 3.6±0.8
This result successfully demonstrates that multiple gene sets of mouse β-Actin related sequences, possibly including many pseudogenes, were detected in mouse genome with Q-PCR. Additionally, a single set of mouse IFNγ was also quantitated as predicted. Altogether, this study validates the sensitivity, specificity, and accuracy of Q-PCR technology and supports its utility for its extensive applications.
1153. Strand Bias in Gene Repair Using SingleStranded Oligonucleotides Charlotte B. Sorensen,1 Anne-Margrethe Krogsdam,2 Karsten Kristiansen,2 Lars Bolund,1 Thomas G. Jensen.1 1 Department of Human Genetics, University of Aarhus, Aarhus, Denmark; 2Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark. Targeted gene conversion is a method developed for site-specific correction of dysfunctional target genes. The method is based on the interaction of either chimeric RNA/DNA or modified single-stranded oligonucleotides with their homologous target genes thereby generating a single mismatched nucleotide base pair. This mismatch is believed to be recognized by the endogenous repair system resulting in excision and substitution of the mismatched base using the oligonucleotide sequence as template. Although the potential of this technique is enormous, it still faces many obstacles such as the relatively low correction efficiencies obtained as well as low reproducibility among and even within laboratories. In order to optimize the efficiency, we are investigating the effect of substituting deoxyribonucleotides in single-stranded oligonucleotides with synthetic DNA-analogues with increased affinity and specificity. As a model system for testing designs of oligonucleotides, we use an episomally expressed beta-galactosidase gene. Oligonuclotides of varying lengths and polarity have been investigated with regard to correction efficiency. Based on the most efficient design, new oligo designs with synthetic DNA-analogues have been made and are being tested. It has previously been shown that antisense oriented singlestranded oligonucleotides results in a higher conversion frequency compared to its sense oriented counterpart. Using standard DNAcontaining oligonucleotides, we have found that this holds true for only one of the beta-galactosidase mutations studied. For the other mutation, the highest efficiency is obtained using the sense oriented oligonucleotide suggesting that limited accessibility due to presence of transcription complexes is not the only cause of the observed strand bias.
Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright ® The American Society of Gene Therapy