Application of semi-quantitative loop-mediated isothermal amplification for gene expression study in Expi293 cells

Abstract

BACKGROUND: Mammalian cell cultures play a key role in the pharmaceutical industry, requiring constant monitoring of the cell conditions during fermentation. In addition to monitoring of the physical-chemical parameters, it is important to evaluate the transcription state of cells, for which gene expression analysis is used. Currently, quantitative reverse transcription polymerase chain reaction (qPCR) is the dominant method. However, the loop-mediated isothermal amplification (LAMP) technique also attracts attention due to its high specificity, sensitivity and reaction rate. LAMP is becoming a promising tool for rapid analysis of gene expression, especially under the conditions of limited biological material or a large volume of samples.

AIM: Development of a technique for semi-quantifying the expression level of target genes in human cell culture Expi293 using LAMP.

MATERIALS AND METHODS: For LAMP, a recombinant large fragment of Bacillus stearothermophilus DNA polymerase (Bst-pol) was obtained, purified, and optimal reaction conditions were determined. SYBR Green I and LUCS13 were used as intercalating dyes. The amplification parameters for different concentrations of the enzyme and the dye were analyzed. Standard SYBR Green I kits were used for qPCR. Both methods were compared when analyzing the expression of IGF1, FGF2 and EIF3i genes in cell lines with an increased expression level of these genes.

RESULTS: It has been shown that using LUCS13 dye provides the classic S-shape of the signal accumulation curve in LAMP, while using SYBR Green I dye causes artifacts. The optimal concentration of Bst-pol was 40 ng/µl. When comparing the two methods, it was found that LAMP has greater sensitivity, allows determining gene expression with an accuracy comparable to qPCR, demonstrating a shorter reaction time (up to 35 minutes).

CONCLUSION: Although qPCR remains the main method for assessing the level of gene expression, LAMP offers a number of advantages that make it an attractive alternative for various biotechnological purposes. Due to its high speed, ease of execution and accessibility, as well as high sensitivity and specificity, LAMP is a valuable technique for rapid analysis of gene expression during cell culture monitoring.

Keywords

real-time polymerase chain reaction / DNA polymerase I / recombinant proteins / loop-mediated isothermal amplification / gene expression

References

[1] Tan E, Chin CSH, Lim ZFS, Ng SK. HEK293 cell line as a platform to produce recombinant proteins and viral vectors. Front Bioeng Biotechnol. 2021;9:796991. doi: 10.3389/fbioe.2021.796991

[2] Tan E., Chin C.S.H., Lim Z.F.S., Ng S.K. HEK293 cell line as a platform to produce recombinant proteins and viral vectors // Front Bioeng Biotechnol. 2021. Vol. 9. P. 796991. doi: 10.3389/fbioe.2021.796991

[3] Demirden SF, Kimiz-Gebologlu I, Oncel SS. Animal cell lines as expression platforms in viral vaccine production: A post Сovid-19 perspective. ACS Omega. 2024;9(15):16904–16926. doi: 10.1021/acsomega.3c10484

[4] Demirden S.F., Kimiz-Gebologlu I., Oncel S.S. Animal cell lines as expression platforms in viral vaccine production: A post Сovid-19 perspective // ACS Omega. 2024. Vol. 9, N. 15. P. 16904–16926. doi: 10.1021/acsomega.3c10484

[5] Merten OW. Development of stable packaging and producer cell lines for the production of AAV vectors. Microorganisms. 2024;12(2):384. doi: 10.3390/microorganisms12020384

[6] Merten O.W. Development of stable packaging and producer cell lines for the production of AAV vectors // Microorganisms. 2024. Vol. 12, N. 2. P. 384. doi: 10.3390/microorganisms12020384

[7] Notomi T, Okayama H, Masubuchi H, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28(12):E63. doi: 10.1093/nar/28.12.e63

[8] Notomi T., Okayama H., Masubuchi H., et al. Loop-mediated isothermal amplification of DNA // Nucleic Acids Res. 2000. Vol. 28, N. 12. P. E63. doi: 10.1093/nar/28.12.e63

[9] Naddaf R, Ben-Assa N, Gefen T, et al. A protocol for simple, rapid, and direct detection of SARS-CoV-2 from clinical samples, using reverse transcribed loop-mediated isothermal amplification (RT-LAMP). Bio Protoc. 2020;10(20):e3789. doi: 10.21769/BioProtoc.3789

[10] Naddaf R., Ben-Assa N., Gefen T., et al. A protocol for simple, rapid, and direct detection of SARS-CoV-2 from clinical samples, using reverse transcribed loop-mediated isothermal amplification (RT-LAMP) // Bio Protoc. 2020. Vol. 10, N. 20. P. e3789. doi: 10.21769/BioProtoc.3789

[11] Park GS, Baek SH, Ku K, et al. Colorimetric RT-LAMP methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Bio Protoc. 2020;10(21):e3804. doi: 10.21769/BioProtoc.3804

[12] Park G.S., Baek S.H., Ku K., et al. Colorimetric RT-LAMP methods to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) // Bio Protoc. 2020. Vol. 10, N. 21. P. e3804. doi: 10.21769/BioProtoc.3804

[13] Anahtar MN, Mcgrath GEG, Rabe BA, et al. Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse transcription loop-mediated isothermal amplification without the need for RNA extraction. Open Forum Infect Dis. 2020;8(2):ofaa631. doi: 10.1093/ofid/ofaa631

[14] Anahtar M.N., Mcgrath G.E.G., Rabe B.A., et al. Clinical assessment and validation of a rapid and sensitive SARS-CoV-2 test using reverse transcription loop-mediated isothermal amplification without the need for RNA extraction // Open Forum Infect Dis. 2020. Vol. 8, N. 2. P. ofaa631. doi: 10.1093/ofid/ofaa631

[15] Takayama I, Nakauchi M, Takahashi H, et al. Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus. J Virol Methods. 2019;267:53–58. doi: 10.1016/j.jviromet.2019.02.010

[16] Takayama I., Nakauchi M., Takahashi H., et al. Development of real-time fluorescent reverse transcription loop-mediated isothermal amplification assay with quenching primer for influenza virus and respiratory syncytial virus // J Virol Methods. 2019. Vol. 267. P. 53–58. doi: 10.1016/j.jviromet.2019.02.010

[17] Huang X, Tang G, Ismail N, Wang X. Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva. EBioMedicine. 2022;75:103736. doi: 10.1016/j.ebiom.2021.103736

[18] Huang X., Tang G., Ismail N., Wang X. Developing RT-LAMP assays for rapid diagnosis of SARS-CoV-2 in saliva // EBioMedicine. 2022. Vol. 75. P. 103736. doi: 10.1016/j.ebiom.2021.103736

[19] Thiessen LD, Neill TM, Mahaffee WF. Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator. PeerJ. 2018;6:e4639. doi: 10.7717/peerj.4639

[20] Thiessen L.D., Neill T.M., Mahaffee W.F. Development of a quantitative loop-mediated isothermal amplification assay for the field detection of Erysiphe necator // PeerJ. 2018. Vol. 6. P. e4639. doi: 10.7717/peerj.4639

[21] Jiang L, Lan X, Ren L, et al. Design of a digital LAMP detection platform based on droplet microfluidic technology. Micromachines. 2023;14(5):1077. doi: 10.3390/mi14051077

[22] Jiang L., Lan X., Ren L., et al. Design of a digital LAMP detection platform based on droplet microfluidic technology // Micromachines (Basel). 2023. Vol. 14, N. 5. P. 1077. doi: 10.3390/mi14051077

[23] Lin X, Huang X, Urmann K, et al. Digital loop-mediated isothermal amplification on a commercial membrane. ACS Sens. 2019;4(1):242–249. doi: 10.1021/acssensors.8b01419

[24] Lin X., Huang X., Urmann K., et al. Digital loop-mediated isothermal amplification on a commercial membrane // ACS Sens. 2019. Vol. 4, N. 1. P. 242–249. doi: 10.1021/acssensors.8b01419

[25] Bobrovsky PA, Grafskaia EN, Kharlampieva DD, et al. Specific activation of growth factors gene expression in the Expi293F human cell line using CRISPR/Cas9-SAM technology leads to increased proliferation. Acta Naturae. 2024;16(3):25–37. EDN: AKHHBB doi: 10.32607/actanaturae.27415

[26] Бобровский П.А., Графская Е.Н., Харлампиева Д.Д., и др. Специфичная активация экспрессии генов ростовых факторов в линии клеток человека Expi293F с помощью технологии CRISPR/Cas9-SAM приводит к повышению пролиферации // Acta Naturae. 2024. Т. 16, № 3. С. 25–37. EDN: AKHHBB doi: 10.32607/actanaturae.27415

[27] Ong J, Evans TC, Tanner N, inventors; New England Biolabs Inc., assignee. DNA polymerases. United States patent US 8993298B1. 2015.

[28] Ong J., Evans T.C., Tanner N., inventors; New England Biolabs Inc., assignee. DNA polymerases. United States Patent US 8993298B1. 2015.

[29] Primerexplorer.jp [Internet]. LAMP primer designing software PrimerExplorer V5 [cited 2024 Aug 23]. Available from: https://primerexplorer.jp/e/

[30] Primerexplorer.jp [Internet]. LAMP primer designing software PrimerExplorer V5. Доступ по ссылке: https://primerexplorer.jp/e/ Дата обращения: 23.08.2024.

[31] Mann HB, Whitney DR. On a test of whether one of two random variables is stochastically larger than the other. Ann Math Statist. 1947;18(1):50–60. doi: 10.1214/aoms/1177730491

[32] Mann H.B., Whitney D.R. On a test of whether one of two random variables is stochastically larger than the other // Ann Math Statist. 1947. Vol. 18, N. 1. P. 50–60. doi: 10.1214/aoms/1177730491

[33] Mcknight P, Najab J. Mann-Whitney U Test. In: Weiner IB, Craighead WE, editors. The Corsini encyclopedia of psychology (4th edition). John Wiley; 2010. doi: 10.1002/9780470479216.corpsy0524

[34] Mcknight P., Najab J. Mann-Whitney U Test. In: Weiner I.B., Craighead W.E., editors. The Corsini encyclopedia of psychology (4th edition). John Wiley, 2010. doi: 10.1002/9780470479216.corpsy0524

[35] Virtanen P, Gommers R, Oliphant TE, et al. SciPy 1.0: fundamental algorithms for scientific computing in Python. Nat Methods. 2020 Mar;17(3):352. doi: 10.1038/s41592-020-0772-5 Erratum for: Nat Methods. 2020;17(3):261–272. doi: 10.1038/s41592-019-0686-2

[36] Virtanen P., Gommers R., Oliphant T.E., et al. SciPy 1.0: fundamental algorithms for scientific computing in Python // Nat Methods. 2020. Vol. 17, N. 3. P. 352. doi: 10.1038/s41592-020-0772-5 Erratum for: Nat Methods. 2020. Vol. 17, N. 3. P. 261–272. doi: 10.1038/s41592-019-0686-2

[37] Neb.com [Internet]. Typical LAMP protocol (M0275) [cited 2024 Aug 23]. Available from: https://www.neb.com/en/protocols/2014/11/21/typical-lamp-protocol-m0275

[38] Neb.com [Internet]. Typical LAMP protocol (M0275). Доступ по ссылке: https://www.neb.com/en/protocols/2014/11/21/typical-lamp-protocol-m0275 Дата обращения: 23.08.2024.

[39] Paik I, Ngo PHT, Shroff R, et al. Improved Bst DNA polymerase variants derived via a machine learning approach. Biochemistry. 2023;62(2):410–418. doi: 10.1021/acs.biochem.1c00451

[40] Paik I., Ngo P.H.T., Shroff R., et al. Improved Bst DNA polymerase variants derived via a machine learning approach // Biochemistry. 2023. Vol. 62, N. 2. P. 410–418. doi: 10.1021/acs.biochem.1c00451

[41] Agustriana E, Nuryana I, Laksmi FA, et al. Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system. Prep Biochem Biotechnol. 2023;53(4):384–393. doi: 10.1080/10826068.2022.2095573

[42] Agustriana E., Nuryana I., Laksmi F.A., et al. Optimized expression of large fragment DNA polymerase I from Geobacillus stearothermophilus in Escherichia coli expression system // Prep Biochem Biotechnol. 2023. Vol. 53, N. 4. P. 384–393. doi: 10.1080/10826068.2022.2095573

[43] Kiefer JR, Mao C, Hansen CJ, et al. Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution. Structure. 1997;5(1):95–108. doi: 10.1016/s0969-2126(97)00169-x

[44] Kiefer J.R., Mao C., Hansen C.J., et al. Crystal structure of a thermostable Bacillus DNA polymerase I large fragment at 2.1 A resolution // Structure. 1997. Vol. 5, N. 1. P. 95–108. doi: 10.1016/s0969-2126(97)00169-x

[45] Quyen TL, Ngo TA, Bang DD, et al. Classification of multiple DNA dyes based on inhibition effects on real-time loop-mediated isothermal amplification (LAMP): Prospect for point of care setting. Front Microbiol. 2019;10:482561. doi: 10.3389/fmicb.2019.02234

[46] Quyen T.L., Ngo T.A., Bang D.D., et al. Classification of multiple DNA dyes based on inhibition effects on real-time loop-mediated isothermal amplification (LAMP): Prospect for point of care setting // Front Microbiol. 2019. Vol. 10. P. 482561. doi: 10.3389/fmicb.2019.02234

[47] Suwatthanarak T, Tanjak P, Chaiboonchoe A, et al. Overexpression of TSPAN8 in consensus molecular subtype 3 colorectal cancer. Exp Mol Pathol. 2024;137:104911. doi: 10.1016/j.yexmp.2024.104911

[48] Suwatthanarak T., Tanjak P., Chaiboonchoe A., et al. Overexpression of TSPAN8 in consensus molecular subtype 3 colorectal cancer // Exp Mol Pathol. 2024. Vol. 137. P. 104911. doi: 10.1016/j.yexmp.2024.104911

[49] Yang J, Chen H, Wang Z, et al. Development of a quantitative loop-mediated isothermal amplification assay for the rapid detection of novel goose parvovirus. Front Microbiol. 2017;8:298309. doi: 10.3389/fmicb.2017.02472

[50] Yang J., Chen H., Wang Z., et al. Development of a quantitative loop-mediated isothermal amplification assay for the rapid detection of novel goose parvovirus // Front Microbiol. 2017. Vol. 8. P. 298309. doi: 10.3389/fmicb.2017.02472

[51] Khan M, Li B, Jiang Y, et al. Evaluation of different PCR-based assays and LAMP method for rapid detection of Phytophthora infestans by targeting the Ypt1 gene. Front Microbiol. 2017;8:1920. doi: 10.3389/fmicb.2017.01920

[52] Khan M., Li B., Jiang Y., et al. Evaluation of different PCR-based assays and LAMP method for rapid detection of Phytophthora infestans by targeting the Ypt1 gene // Front Microbiol. 2017. Vol. 8. P. 1920. doi: 10.3389/fmicb.2017.01920

[53] Cao Z, Zhang K, Yin D, et al. Clinical validation of visual LAMP and qLAMP assays for the rapid detection of Toxoplasma gondii. Front Cell Infect Microbiol. 2022;12:1024690. doi: 10.3389/fcimb.2022.1024690

[54] Cao Z., Zhang K., Yin D., et al. Clinical validation of visual LAMP and qLAMP assays for the rapid detection of Toxoplasma gondii // Front Cell Infect Microbiol. 2022. Vol. 12. P. 1024690. doi: 10.3389/fcimb.2022.1024690

[55] Svec D, Tichopad A, Novosadova V, et al. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif. 2015;3:9–16. doi: 10.1016/j.bdq.2015.01.005

[56] Svec D., Tichopad A., Novosadova V., et al. How good is a PCR efficiency estimate: Recommendations for precise and robust qPCR efficiency assessments // Biomol Detect Quantif. 2015. Vol. 3. P. 9–16. doi: 10.1016/j.bdq.2015.01.005

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