Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cloning of Recombinant Adenoviral Vectors Containing the mCherry Gene
2.2. Animal Experiments
2.2.1. Acute Experiments
2.2.2. Chronic Experiments
2.3. Confirmation of Gene and Protein Expression by Infection of rAdV Vectors In Vivo
2.4. DNA Extraction and Detection of Transgenes Using Three Different PCR Methods
2.5. Semi-Quantitative PCR (sqPCR)
2.6. Real-Time Quantitative PCR (qPCR)
2.7. Droplet Digital PCR (ddPCR)
2.8. Statistics
3. Results
3.1. The mCherry Gene and Protein Were Sufficiently Expressed Both In Vitro and In Vivo
3.2. The Three PCR Methods Showed Each Characteristic and Could Detect Transgene Fragments in Acute Experiments
3.3. ddPCR on Chronic Experiments Showed a Possibility of Detecting Transgenes Repeatedly
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Vector | Gene Therapy Clinical Trials | |
---|---|---|
Number | % | |
Adenovirus | 541 | 18.5 |
Retrovirus | 514 | 17.5 |
Naked/Plasmid DNA | 452 | 15.4 |
Lentivirus | 278 | 9.5 |
Adeno-associated virus | 238 | 8.1 |
Vaccinia virus | 133 | 4.5 |
Lipofection | 119 | 4.1 |
Others | 774 | 26.4 |
Total | 2930 | 100 |
Methods | Targets | Sequences | Predicted Size (bp) | |
---|---|---|---|---|
sqPCR | mCherry gene body | Forward | CACGAGTTCGAGATCGAGGG | 234 |
Reverse | GCCGTCCTCGAAGTTCATCA | |||
sqPCR | CMV promoter | Forward | CACGCCCATTGATGTACTGC | 247 |
Reverse | ACGCCAATAGGGACTTTCCA | |||
qPCR, ddPCR: Taq man probe assay | mCherry gene body | Forward | GGCACCAACTTCCCCTCC | 115 |
Probe | 56FAM/CATGGTCTT/ZEN/CTTCTGCAT/3IABkFQ | |||
Reverse | TCTGCTTGATCTCGCCCTTC | |||
qPCR: SYBR green assay | 18s rRNA | Forward | AGTCCCTGCCCTTTGTACACA | 70 |
Reverse | CGATCCGAGGGCCTCACTA |
Group | Mouse No. | Copy/μL of Transgene | ||
---|---|---|---|---|
Blood Cell Fraction-DNA | Plasma-cfDNA | Stool-DNA | ||
Con. | 1 | 0.0 | 0.0 | 0.0 |
2 | 0.0 | 0.0 | 0.0 | |
3 | 0.0 | 0.0 | 0.0 | |
4 | 0.0 | 0.0 | 0.0 | |
5 | 0.0 | 0.0 | 0.0 | |
6 | 0.0 | 0.0 | 0.0 | |
Median | 0.0 | 0.0 | 0.0 | |
IV, rAdV mCherry | 1 | 2528.2 | 33.4 | 3.7 |
2 | 390.3 | 3.3 | 0.0 | |
3 | 1808.4 | 8.5 | 1.6 | |
4 | 217.9 | 5.1 | 0.0 | |
5 | 230.4 | 4.9 | 0.9 | |
6 | 26.2 | 5.5 | 0.8 | |
7 | 63.1 | 4.0 | 2.6 | |
Median | 230.4 a, b | 5.1 a, b | 0.9 a, b | |
LM, rAdV mCherry | 1 | 56.2 | 0.0 | 1.8 |
2 | 11.8 | 0.0 | 0.0 | |
3 | 16.5 | 0.0 | 0.0 | |
4 | 30.7 | 0.0 | 0.0 | |
5 | 15.9 | 0.0 | 0.0 | |
6 | 31.7 | 0.0 | 0.0 | |
7 | 28.3 | 0.0 | 0.0 | |
Median | 28.3 c | 0.0 | 0.0 |
Group | Mouse No. | Copy/μL of Transgene | ||
---|---|---|---|---|
Blood Cell Fraction-DNA | Plasma-cfDNA | Stool-DNA | ||
Con. | 1 | 0.0 | 0.0 | 0.0 |
2 | 0.0 | 0.0 | 0.0 | |
3 | 0.0 | 0.8 | 0.7 | |
4 | 0.0 | 0.0 | 0.0 | |
5 | 0.6 | 0.0 | 0.0 | |
6 | 0.0 | 0.8 | 0.0 | |
Median | 0.0 | 0.0 | 0.0 | |
IV, rAdV mCherry | 1 | 4460.0 | 19.2 | 2.3 |
2 | 620.7 | 0.7 | 2.2 | |
3 | 2873.3 | 6.0 | 5.5 | |
4 | 276.7 | 4.3 | 0.0 | |
5 | 190.0 | 2.3 | 0.0 | |
6 | 13.7 | 1.9 | 0.8 | |
7 | 42.7 | 2.5 | 1.5 | |
Median | 276.7 a, b | 2.5 a, b | 1.5 a | |
LM, rAdV mCherry | 1 | 34.0 | 0.0 | 0.7 |
2 | 5.5 | 0.0 | 0.0 | |
3 | 4.5 | 3.0 | 0.7 | |
4 | 12.9 | 1.4 | 0.0 | |
5 | 3.8 | 0.0 | 0.7 | |
6 | 16.5 | 0.0 | 1.4 | |
7 | 11.5 | 0.0 | 1.5 | |
Median | 11.5 c | 0.0 | 0.7 d |
Mouse No. | Copy/μL of Transgene | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Pre | 1 day | 3 days | 5 days | 7 days | 9 days | 11 days | 13 days | 15 days | ||
IV, rAdV mCherry | 1 | 0.0 | 817.0 | 1398.0 | 48.0 | 4.3 | 1.4 | 0.0 | 0.8 | 0.0 |
2 | 0.0 | 890.0 | 209.0 | 25.0 | 2.4 | 0.0 | 0.7 | 3.4 | 0.7 | |
3 | 0.8 | 1108.0 | 796.0 | 10.3 | 0.9 | 0.0 | 0.0 | 10.4 | 0.0 | |
4 | 2.0 | 1422.0 | 298.0 | 13.7 | 1.4 | 2.7 | 1.7 | 0.0 | 0.0 | |
5 | 0.7 | 1900.0 | 1132.0 | 62.0 | 10.5 | 1.4 | 1.6 | 1.5 | 0.8 | |
6 | 0.0 | 4800.0 | 1261.0 | 39.0 | 7.1 | 2.0 | 0.7 | 0.9 | 1.5 | |
Median | 0.4 | 1265.0 | 964.0 | 32.0 | 3.4 | 1.4 | 0.7 | 1.2 | 0.4 | |
p- values vs Pre | 0.0002 | 0.0004 | 0.0063 | 0.0911 | 0.4181 | 0.5886 | 0.2992 | 0.6177 | ||
LM, rAdV mCherry | 1 | 0.0 | 70.0 | 66.0 | 7.3 | 0.7 | 0.8 | 0.0 | 0.0 | 0.0 |
2 | 0.9 | 149.0 | 24.0 | 5.0 | 1.6 | 0.7 | 0.0 | 0.0 | 0.0 | |
3 | 0.0 | 19.0 | 7.2 | 4.2 | 0.7 | 0.0 | 0.0 | 0.0 | 0.0 | |
4 | 0.0 | 10.4 | 4.6 | 0.8 | 0.8 | 0.7 | 0.0 | 0.0 | 0.0 | |
5 | 0.0 | 8.6 | 9.0 | 0.0 | 0.8 | 0.8 | 0.0 | 1.5 | 0.0 | |
6 | 0.0 | 5.2 | 27.0 | 4.1 | 0.0 | 0.7 | 0.0 | 0.0 | 0.0 | |
Median | 0.0 | 14.7 | 16.5 | 4.2 | 0.8 | 0.7 | 0.0 | 0.0 | 0.0 | |
p- values vs Pre | 0.0012 | 0.0012 | 0.0666 | 0.2576 | 0.2902 | 0.5904 | 0.7753 | 0.5907 |
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Sugasawa, T.; Aoki, K.; Watanabe, K.; Yanazawa, K.; Natsume, T.; Takemasa, T.; Yamaguchi, K.; Takeuchi, Y.; Aita, Y.; Yahagi, N.; et al. Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR. Genes 2019, 10, 436. https://doi.org/10.3390/genes10060436
Sugasawa T, Aoki K, Watanabe K, Yanazawa K, Natsume T, Takemasa T, Yamaguchi K, Takeuchi Y, Aita Y, Yahagi N, et al. Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR. Genes. 2019; 10(6):436. https://doi.org/10.3390/genes10060436
Chicago/Turabian StyleSugasawa, Takehito, Kai Aoki, Koichi Watanabe, Koki Yanazawa, Tohru Natsume, Tohru Takemasa, Kaori Yamaguchi, Yoshinori Takeuchi, Yuichi Aita, Naoya Yahagi, and et al. 2019. "Detection of Transgenes in Gene Delivery Model Mice by Adenoviral Vector Using ddPCR" Genes 10, no. 6: 436. https://doi.org/10.3390/genes10060436