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An Efficient Probe-Based Quantitative PCR Assay Targeting Human-Specific DNA in ST6GALNAC3 for the Quantification of Human Cells in Preclinical Animal Models

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Abstract

Precise quantification of human cells in preclinical animal models by a sensitive and specific approach is warranted. The probe-based quantitative PCR (qPCR) assay as a sensitive and swift approach is suitable for the quantification of human cells by targeting human-specific DNA sequences. In this study, we developed an efficient qPCR assay targeting human-specific DNA in ST6GALNAC3 (termed ST6GAL-qPCR) for the quantification of human cells in preclinical animal models. ST6GAL-qPCR probe was synthesized with FAM and non-fluorescent quencher-minor groove binder conjugated to the 5′ and 3′ end of the probe, respectively. Genomic DNA from human, rhesus monkeys, cynomolgus monkeys, New Zealand White rabbits, SD rats, C57BL/6, and BALB/c mice were utilized for analyzing the specificity and sensitivity of the ST6GAL-qPCR assay. The ST6GAL-qPCR assay targeted human-specific DNA was cloned to pUCM-T vector and released by EcoR I/Hind III digestion for generating a calibration curve. Cell mixing experiment was performed to validate the ST6GAL-qPCR assay by analysis of 0.1%, 0.01%, and 0.001% of human leukocytes mixed with murine thymocytes. The ST6GAL-qPCR assay detected human DNA rather than DNA from the tested animal species. The amplification efficiency of the ST6GAL-qPCR assay was 93% and the linearity of calibration curve was R2 = 0.999. The ST6GAL-qPCR assay detected as low as 5 copies of human-specific DNA and is efficient to specially amplify as low as 30-pg human DNA in the presence of 1 μg of DNA from the tested species, respectively. The ST6GAL-qPCR assay was able to quantify as low as 0.01% of human leukocytes within murine thymocytes. This ST6GAL-qPCR assay can be used as an efficient approach for the quantification of human cells in preclinical animal models.

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References

  1. El-Kadiry, A. E., Rafei, M., & Shammaa, R. (2021). Cell therapy: Types, regulation, and clinical benefits. Front Med (Lausanne), 8, 756029.

    Article  PubMed  Google Scholar 

  2. Bastien, J. P., Minguy, A., Dave, V., & Roy, D. C. (2019). Cellular therapy approaches harnessing the power of the immune system for personalized cancer treatment. Seminars in Immunology, 42, 101306.

    Article  CAS  PubMed  Google Scholar 

  3. Wang, L. L., Janes, M. E., Kumbhojkar, N., Kapate, N., Clegg, J. R., Prakash, S., Heavey, M. K., Zhao, Z., Anselmo, A. C., & Mitragotri, S. (2021). Cell therapies in the clinic. Bioeng Transl Med 6(2), e10214.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Campbell, A., Brieva, T., Raviv, L., Rowley, J., Niss, K., Brandwein, H., Oh, S., & Karnieli, O. (2015). Concise Review: Process development considerations for cell therapy. Stem Cells Transl Med 4(10), 1155-1163.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Osada, N. (2015). Genetic diversity in humans and non-human primates and its evolutionary consequences. Genes and Genetic Systems, 90(3), 133–145.

    Article  CAS  PubMed  Google Scholar 

  6. Harding, J., Roberts, R. M., & Mirochnitchenko, O. (2013). Large animal models for stem cell therapy. Stem Cell Research and Therapy, 4(2), 23.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Yamamoto, S., Ding, N., Matsumoto, S. I., & Hirabayashi, H. (2021). Highly specific, quantitative polymerase chain reaction probe for the quantification of human cells in cynomolgus monkeys. Drug Metab Pharmacokinet 36, 100359.

    Article  CAS  PubMed  Google Scholar 

  8. Song, P., Xie, Z., Guo, L., Wang, C., Xie, W., & Wu, Y. (2012). Human genome-specific real-time PCR method for sensitive detection and reproducible quantitation of human cells in mice. Stem Cell Rev Rep 8(4),1155–1162.

    Article  CAS  PubMed  Google Scholar 

  9. Kline, M. C., Romsos, E. L., & Duewer, D. L. (2016). Evaluating digital PCR for the quantification of human genomic DNA: Accessible amplifiable targets. Analytical Chemistry, 88(4), 2132–2139.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Prigent, J., Herrero, A., Ambroise, J., Smets, F., Deblandre, G. A., & Sokal, E. M. (2015). Human progenitor cell quantification after xenotransplantation in rat and mouse models by a sensitive qPCR Assay. Cell Transplant 24(8), 1639–1652.

    Article  PubMed  Google Scholar 

  11. Funakoshi, K, Bagheri, M., Zhou, M., Suzuki, R., Abe, H., & Akashi, H. (2017). Highly sensitive and specific Alu-based quantification of human cells among rodent cells. Scientific reports 7(1), 13202.

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  12. Suntsova, M. V., & Buzdin, A. A. (2020). Differences between human and chimpanzee genomes and their implications in gene expression, protein functions and biochemical properties of the two species. BMC Genomics, 21(Suppl 7), 535.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Lander, E.S., Linton, L.M., Birren, B., Nusbaum, C., Zody, M. C. & ... (2001). Initial sequencing and analysis of the human genome. Nature 409(6822), 860–921.

    Article  CAS  PubMed  ADS  Google Scholar 

  14. McBlane, J. W., Phul, P., & Sharpe, M. (2018). Preclinical development of cell-based products: A European regulatory science perspective. Pharmaceutical Research, 35(8), 165.

    Article  PubMed  PubMed Central  Google Scholar 

  15. Smithson, M., Irwin, R., Williams, G., Alexander, K. L., Smythies, L. E., Nearing, M., McLeod, M. C., Al Diffalha, S., Bellis, S. L., & Hardiman, K. M. (2022). Sialyltransferase ST6GAL-1 mediates resistance to chemoradiation in rectal cancer. J Biol Chem 298(3), 101594.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Liu, X., Liu, Y., Ma, Y., Gong, Y., Liu, Q., Sun, W., & Guo, H. (2021). Establishment of patient-specific induced pluripotent stem cell line SDUBMSi009-A from a patient with X-linked Lowe syndrome. Stem Cell Res 51, 102171.

    Article  CAS  PubMed  Google Scholar 

  17. Kent, W. J. (2002). BLAT—The BLAST-like alignment tool. Genome Research, 12(4), 656–664.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Chimpanzee, S., & Analysis, C. (2005). Initial sequence of the chimpanzee genome and comparison with the human genome. Nature, 437(7055), 69–87.

    Article  Google Scholar 

  19. Piovesan, A., Pelleri, M.C., Antonaros, F., Strippoli, P., Caracausi, M., & Vitale, L. (2019). On the length, weight and GC content of the human genome. BMC Res Notes 12(1), 106.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Soejima, M., Hiroshige, K., Yoshimoto, J., & Koda, Y. (2012). Selective quantification of human DNA by real-time PCR of FOXP2. Forensic Sci Int Genet 6(4), 447–451.

    Article  CAS  PubMed  Google Scholar 

  21. Zong, C., Lu, S., Chapman, A. R., & Xie, X. S. (2012). Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338(6114), 1622–1626.

    Article  CAS  PubMed  PubMed Central  ADS  Google Scholar 

  22. Hiroshige, K., Soejima, M., Nishioka, T., Kamimura, S., & Koda, Y. (2009). Simple and sensitive method for identification of human DNA by allele-specific polymerase chain reaction of FOXP2. J Forensic Sci 54(4), 857–861.

    Article  CAS  Google Scholar 

  23. Atkins, J. T., George, G. C., Hess, K., Marcelo-Lewis, K. L., Yuan, Y., Borthakur, G., Khozin, S., LoRusso, P., & Hong, D. S. (2020) Pre-clinical animal models are poor predictors of human toxicities in phase 1 oncology clinical trials. Br J Cancer 123(10), 1496–1501.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Van Norman, G. A. (2019). Limitations of animal studies for predicting toxicity in clinical trials: Is it time to rethink our current approach? JACC Basic to Translational Science, 4(7), 845–854.

    Article  PubMed  PubMed Central  ADS  Google Scholar 

  25. Chua, D., Low, Z. S., Cheam, G. X., Ng, A. S., & Tan, N. S. (2022). Utility of human relevant preclinical animal models in navigating NAFLD to MAFLD paradigm. Int J Mol Sci 23(23).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Shigeto, J., Ichiki, T., Nii, T., Konno, K., Nakanishi, Y., & Sugiyama, D. (2018). Preclinical toxicity studies for regenerative medicine in Japan. Clin Ther 40(11), 1813–1822.

    Article  Google Scholar 

  27. Walker, J. A., Kilroy, G. E., Xing, J., Shewale, J., Sinha, S. K., & Batzer, M. A. (2003). Human DNA quantitation using Alu element-based polymerase chain reaction. Anal Biochem 315(1), 122–128.

    Article  CAS  PubMed  Google Scholar 

  28. Walker, J. A., Hedges, D. J., Perodeau, B. P., Landry, K. E., Stoilova, N., Laborde, M. E., Shewale, J., Sinha, S. K., & Batzer, M. A. (2005). Multiplex polymerase chain reaction for simultaneous quantitation of human nuclear, mitochondrial, and male Y-chromosome DNA: application in human identification. Anal Biochem 337(1), 89–97.

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grant No. 82171791), Xuzhou Science and Technology Program (KC20089), the Youth Innovation Team Grant and the Starting Grant by Xuzhou Medical University (Grant No. D2018009), the Postgraduate Research and Practice Innovation Program of Jiangsu Province, China (KYCX21_2638 and KYCX22_2875), Jiangsu Training Program of Innovation and Entrepreneurship for Undergraduates (202210313005Z), and the Special Science and Technology Project on Life and Health by Nanjing Municipal Bureau of Science and Technology (202205009).

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Author notes

  1. Jinfeng Ren, Ke Liu, Lang Hu, and Ruoning Yang have contributed equally and should be considered as joint first author.

Authors and Affiliations

  1. Jiangsu Key Laboratory of Immunity and Metabolism, Department of Pathogenic Biology and Immunology, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China

    Jinfeng Ren, Lang Hu, Yuting Liu, Siyu Wang, Xinzhu Chen, Hui Wang & Hui Li

  2. National Experimental Demonstration Center for Basic Medicine Education, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China

    Jinfeng Ren, Lang Hu, Yuting Liu, Siyu Wang, Xinzhu Chen, Hui Wang & Hui Li

  3. Department of Gastroenterology, The Affiliated Hospital of Xuzhou Medical University, Xuzhou, 221000, Jiangsu, China

    Ke Liu

  4. Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical University, Xuzhou, 221004, Jiangsu, China

    Ruoning Yang, Luyao Jing, Tiantian Liu, Bin Hu & Hui Li

  5. General Clinical Research Center, Nanjing First Hospital, Nanjing Medical University, Nanjing, China

    Shuli Zhao

  6. Jiangsu Tripod Preclinical Research Laboratories Inc, Nanjing, China

    Xuefeng Zhang

  7. Department of Pathogenic Biology and Immunology, Xuzhou Medical University, 209 Tongshan Road, Xuzhou, 221004, China

    Hui Li

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  1. Jinfeng Ren
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Contributions

JR, KL, LH, RY, YL, SW, XC, LJ, TL, and BH performed the experiments. JR, KL, and LH performed data analysis. SZ and XZ contributed the collection of human and monkey samples. HL and HW conceived and supervised the study. HL and HW prepared the figures and wrote the manuscript.

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Correspondence to Hui Wang or Hui Li.

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Supplementary Information

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Supplementary file1 (DOCX 14 kb)

12033_2024_1115_MOESM2_ESM.pdf

Supplementary file2 (PDF 274 kb) DNA alignment of human-specific DNA in ST6GALNAC3 with ST6GALNAC3 genomic DNA of rhesus and cynomolgus monkeys. A 1460 bp of DNA fragment in human ST6GALNAC3 was aligned with rhesus (A) and cynomolgus (B) ST6GALNAC3 genomic DNA

12033_2024_1115_MOESM3_ESM.pdf

Supplementary file3 (PDF 475 kb) Validation of the ST6GAL-qPCR products by DNA sequencing. The PCR products of the ST6GAL-qPCR was cloned to pUCM-T vectors and subsequently analyzed by DNA sequencing

12033_2024_1115_MOESM4_ESM.jpg

Supplementary file4 (JPG 77 kb) Analysis of human genomic DNA by the ST6GAL-qPCR assay. A representative amplification plot of the ST6GAL-qPCR analysis of genomic DNA (1–10 ng) of 48 individuals

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Ren, J., Liu, K., Hu, L. et al. An Efficient Probe-Based Quantitative PCR Assay Targeting Human-Specific DNA in ST6GALNAC3 for the Quantification of Human Cells in Preclinical Animal Models. Mol Biotechnol (2024). https://doi.org/10.1007/s12033-024-01115-8

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