regmedjournalРегенерация органов и тканейРегенерация органов и тканей2949-5938Общество регенеративной медицины10.60043/2949-5938-2024-1-16-28regmedjournal-49Research ArticleОБЗОРЫ И КОММЕНТАРИИREVIEWS AND COMMENTSМетод Golden Gate в биологии и медицинеGolden gate method in biology and medicineАнтипинаМ. И.AntipinaM. I.

Антипина Мария Игоревна — инженер-исследователь НИЛ трансляционных исследований ОНК «Институт медицины и наук о жизни (МЕДБИО)»

ул. Александра Невского, д. 14, 236041, Калининград

Maria I. Antipina — Research Engineer at the Translational Research Laboratory of the Institute of Medicine and Life Sciences (MEDBIO)

Alexander Nevsky Str., 14, 236041, Kaliningrad

antipinaria@gmail.comЛиВ. А.LiV. A.

Ли Владислав Артурович — студент 4-го курса специалитета по направлению «Биоинженерия и биоинформатика» ОНК «Институт медицины и наук о жизни (МЕДБИО)»

ул. Александра Невского, д. 14, 236041, Калининград

Vladislav A. Li — 4th-year student specializing in Bioengineering and Bioinformatics at the Institute of Medicine and Life Sciences (MEDBIO)

Alexander Nevsky Str., 14, 236041, Kaliningrad

ПоповаЕ. Е.PopovaE. E.

Попова Елизавета Евгеньевна — студентка 4-го курса специалитета по направлению «Биоинженерия и биоинформатика» ОНК «Институт медицины и наук о жизни (МЕДБИО)»

ул. Александра Невского, д. 14, 236041, Калининград

Elizaveta E. Popova  — 4th-year student specializing in Bioengineering and Bioinformatics at the Institute of Medicine and Life Sciences (MEDBIO)

Alexander Nevsky Str., 14, 236041, Kaliningrad

СеминаЕ. В.SeminaE. V.

Семина Екатерина Владимировна — д.б.н., зав. лаб. НИЛ трансляционных исследований ОНК «Институт медицины и наук о жизни (МЕДБИО)»

ул. Александра Невского, д. 14, 236041, Калининград

Ekaterina V. Semina  — Dr. Sci. (Biology), Head of the Translational Research Laboratory at the Institute of Medicine and Life Sciences (MEDBIO)

Alexander Nevsky Str., 14, 236041, Kaliningrad

ФГАОУ ВО «Балтийский федеральный университет им. И. Канта»РоссияImmanuel Kant Baltic Federal UniversityRussian Federation202412102024211628Copyright © Антипина М.И., Ли В.А., Попова Е.Е., Семина Е.В., 20242024Антипина М.И., Ли В.А., Попова Е.Е., Семина Е.В.Antipina M.I., Li V.A., Popova E.E., Semina E.V.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.regmed-journal.ru/jour/article/view/49

Целью данного обзора было описать и сравнить методы молекулярного клонирования для сборки генетических конструкций. Генетическая терапия — одна из активно развивающихся отраслей современной медицины, поэтому особое внимание в данном обзоре уделено таким параметрам, как быстрота, точность и эффективность клонирования, так как они являются критическими факторами при создании генно-терапевтических средств. Особое внимание уделено методике Golden Gate, которая основана на использовании эндонуклеаз рестрикции типа IIS, поскольку такой подход упрощает процесс клонирования и повышает его эффективность за счет стандартизированного дизайна и минимального набора ферментов. Наряду с Golden Gate в обзоре также обсуждаются такие методы молекулярного клонирования, как Gateway и Gibson, с точки зрения перспектив их использования для решения фундаментальных и прикладных задач регенеративной медицины.

The aim of this review was to describe and compare molecular cloning methods for assembling genetic constructs. Genetic therapy is one of the rapidly developing fields of modern medicine, so special attention in this review is paid to parameters such as speed, accuracy, and efficiency of cloning, as these are critical factors in creating gene therapy agents. Special attention is given to the Golden Gate method, which is based on the use of type IIS restriction endonucleases, as this approach simplifies the cloning process and increases its efficiency through standardized design and a minimal set of enzymes. Alongside Golden Gate, the review also discusses more innovative molecular cloning methods, Gateway and Gibson, in terms of their potential use for addressing fundamental and applied challenges in regenerative medicine.

молекулярное клонированиеGolden Gateклонирование Gatewayклонирование Gibsonmolecular cloningGolden GateGateway cloningGibson AssemblyИсследование поддержано из средств программы стратегического академического лидерства «Приоритет 2030» БФУ им. КантаThis research was supported from the Russian Federal Academic Leadership Program Priority 2030 at the Immanuel Kant Baltic Federal University.ReferencesWeber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS One. 2011;6(2):e16765. DOI: 10.1371/journal.pone.0016765Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLOS One. 2011;6(2):e16765. DOI: 10.1371/journal.pone.0016765A Quick Overview Of Molecular Cloning. https://www.goldbio.com/articles/article/cloning-overview [Accessed March 31, 2024].A Quick Overview Of Molecular Cloning. https://www.goldbio.com/articles/article/cloning-overview [Accessed March 31, 2024].Williams SA, Slatko BE, McCarrey JR. Laboratory investigations in molecular biology. Jones and Bartlett Publishers, 2007.Williams SA, Slatko BE, McCarrey JR. Laboratory investigations in molecular biology. Jones and Bartlett Publishers, 2007.Struhl K. Subcloning of DNA fragments. Current Protocols in Molecular Biology. 1991; 13(1):3.16. DOI: 10.1002/0471142727.mb0316s13Struhl K. Subcloning of DNA fragments. Current Protocols in Molecular Biology. 1991; 13(1):3.16. DOI: 10.1002/0471142727.mb0316s13Shetty RP, Endy D, Knight TFJr. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2008;2(1):5. DOI: 10.1186/1754-1611-2-5Shetty RP, Endy D, Knight TFJr. Engineering BioBrick vectors from BioBrick parts. Journal of Biological Engineering. 2008;2(1):5. DOI: 10.1186/1754-1611-2-5Sleight SC, Bartley BA, Lieviant, JA, and Sauro HM. In-Fusion BioBrick assembly and reengineering. Nucleic Acids Research. 2010;38(8):2624–2636. DOI: 10.1093/nar/gkq179Sleight SC, Bartley BA, Lieviant, JA, and Sauro HM. In-Fusion BioBrick assembly and reengineering. Nucleic Acids Research. 2010;38(8):2624–2636. DOI: 10.1093/nar/gkq179Hoseini S, Sauer MG. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering. Journal of Biological Engineering. 2015;9(1):2. DOI: 10.1186/1754-1611-9-2Hoseini S, Sauer MG. Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering. Journal of Biological Engineering. 2015;9(1):2. DOI: 10.1186/1754-1611-9-2Motohashi K. A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products. Scientific Reports. 2019;9(1):6417. DOI: 10.1038/s41598-019-42868-6Motohashi K. A novel series of high-efficiency vectors for TA cloning and blunt-end cloning of PCR products. Scientific Reports. 2019;9(1):6417. DOI: 10.1038/s41598-019-42868-6Zhou MY, Gomez-Sanchez CE. Universal TA Cloning. Current Issues in Molecular Biology. 2000;2:1–7. DOI: 10.21775/cimb.002.001Zhou MY, Gomez-Sanchez CE. Universal TA Cloning. Current Issues in Molecular Biology. 2000;2:1–7. DOI: 10.21775/cimb.002.001Plasmid 101: TOPO Cloning https://blog.addgene.org/plasmids-101-topo-cloning [Accessed April 10, 2024].Plasmid 101: TOPO Cloning https://blog.addgene.org/plasmids-101-topo-cloning [Accessed April 10, 2024].Shuman S. Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific. Proceedings of the National Academy of Sciences. 1991;88(22):10104–10108. DOI: 10.1073/pnas.88.22.10104Shuman S. Recombination mediated by vaccinia virus DNA topoisomerase I in Escherichia coli is sequence specific. Proceedings of the National Academy of Sciences. 1991;88(22):10104–10108. DOI: 10.1073/pnas.88.22.10104Shuman S. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. Journal of Biological Chemistry. 1994;269(51):32678–32684. DOI: 10.1016/s0021-9258(18)31688-0Shuman S. Novel approach to molecular cloning and polynucleotide synthesis using vaccinia DNA topoisomerase. Journal of Biological Chemistry. 1994;269(51):32678–32684. DOI: 10.1016/s0021-9258(18)31688-0Reece-Hoyes JS, Walhout AJM. Gateway recombinational cloning. Cold Spring Harbor Protocols. 2018;2018(1):pdb.top094912. DOI: 10.1101/pdb.top094912Reece-Hoyes JS, Walhout AJM. Gateway recombinational cloning. Cold Spring Harbor Protocols. 2018;2018(1):pdb.top094912. DOI: 10.1101/pdb.top094912Chin CF, Chee JY. Gateway cloning technology: Advantages and drawbacks. Cloning & Transgenesis. 2015;04(01):1-3. DOI: 10.4172/2168-9849.1000138Chin CF, Chee JY. Gateway cloning technology: Advantages and drawbacks. Cloning & Transgenesis. 2015;04(01):1-3. DOI: 10.4172/2168-9849.1000138Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CAIII, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 2009;6(5):343– 345. DOI: 10.1038/nmeth.1318Gibson DG, Young L, Chuang RY, Venter JC, Hutchison CAIII, Smith HO. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods. 2009;6(5):343– 345. DOI: 10.1038/nmeth.1318Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science. 2010;329(5987):52–56. DOI: 10.1126/science.1190719Gibson DG, Glass JI, Lartigue C, Noskov VN, Chuang RY, Algire MA, et al. Creation of a bacterial cell controlled by a chemically synthesized genome. Science. 2010;329(5987):52–56. DOI: 10.1126/science.1190719Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLOS One. 2008;3(11):e3647. DOI: 10.1371/journal.pone.0003647Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLOS One. 2008;3(11):e3647. DOI: 10.1371/journal.pone.0003647Engler C, Marillonnet S. Combinatorial DNA assembly using Golden Gate cloning. In Synthetic Biology. Humana Press. 2013;1073:141-156. DOI: 10.1007/978-1-62703-625-2_12Engler C, Marillonnet S. Combinatorial DNA assembly using Golden Gate cloning. In Synthetic Biology. Humana Press. 2013;1073:141-156. DOI: 10.1007/978-1-62703-625-2_12Chiasson D, Giménez-Oya V, Bircheneder M, Bachmaier S, Studtrucker T, Ryan J, et al. A unified multi-kingdom Golden Gate cloning platform. Scientific Reports. 2019;9(1):10131. DOI: 10.1038/s41598-019-46171-2Chiasson D, Giménez-Oya V, Bircheneder M, Bachmaier S, Studtrucker T, Ryan J, et al. A unified multi-kingdom Golden Gate cloning platform. Scientific Reports. 2019;9(1):10131. DOI: 10.1038/s41598-019-46171-2Potapov V, Ong JL, Kucera RB, Langhors BW, Bilotti K, Pryor JM, et al. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly. ACS Synthetic Biology. 2018;7(11):2665–2674. DOI: 10.1021/acssynbio.8b00333Potapov V, Ong JL, Kucera RB, Langhors BW, Bilotti K, Pryor JM, et al. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly. ACS Synthetic Biology. 2018;7(11):2665–2674. DOI: 10.1021/acssynbio.8b00333Plasmid 101: Golden Gate Cloning https://blog.addgene.org/plasmids-101-golden-gate-cloning [Accessed April 17, 2024].Plasmid 101: Golden Gate Cloning https://blog.addgene.org/plasmids-101-golden-gate-cloning [Accessed April 17, 2024].New England BioLabs: Golden Gate Assembly https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly [Accessed April 17, 2024].New England BioLabs: Golden Gate Assembly https://international.neb.com/applications/cloning-and-synthetic-biology/dna-assembly-and-cloning/golden-gate-assembly [Accessed April 17, 2024].Kues WA, Kumar D, Selokar NL, Talluri TR. Applications of genome editing tools in stem cells towards regenerative medicine: An update. Current Stem Cell Research & Therapy 2022;17(3):267–279. DOI: 10.2174/1574888X16666211124095527Kues WA, Kumar D, Selokar NL, Talluri TR. Applications of genome editing tools in stem cells towards regenerative medicine: An update. Current Stem Cell Research & Therapy 2022;17(3):267–279. DOI: 10.2174/1574888X16666211124095527Barker JC, Barker AD, Bills J, Huang J, Wight-Carter M, Delgado I, et al. Genome Editing of Mouse Fibroblasts by Homologous Recombination for Sustained Secretion of PDGF-B and Augmentation of Wound Healing. Plastic and Reconstructive Surgery. 2014;134(3):389e–401e. doi: 10.1097/PRS.0000000000000427Barker JC, Barker AD, Bills J, Huang J, Wight-Carter M, Delgado I, et al. Genome Editing of Mouse Fibroblasts by Homologous Recombination for Sustained Secretion of PDGF-B and Augmentation of Wound Healing. Plastic and Reconstructive Surgery. 2014;134(3):389e–401e. doi: 10.1097/PRS.0000000000000427Niu W, Zang T, Zou Y, Fang S, Smith DK, Bachoo R, et al. In vivo reprogramming of astrocytes to neuroblasts in the adult brain. Nature Cell Biology. 2013;15(10):1164–1175. doi: 10.1038/ncb2843Niu W, Zang T, Zou Y, Fang S, Smith DK, Bachoo R, et al. In vivo reprogramming of astrocytes to neuroblasts in the adult brain. Nature Cell Biology. 2013;15(10):1164–1175. doi: 10.1038/ncb2843György B, Lööv C, Zaborowski MP, Takeda S, Kleinstiver BP, Commins C, et al. CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease. Molecular Therapy — Nucleic Acids. 2018;11:429–440. doi: 10.1016/j.omtn.2018.03.007György B, Lööv C, Zaborowski MP, Takeda S, Kleinstiver BP, Commins C, et al. CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease. Molecular Therapy — Nucleic Acids. 2018;11:429–440. doi: 10.1016/j.omtn.2018.03.007Guo Z, Zhang L, Wu Z, Chen Y, Wang F, Chen G. In Vivo Direct Reprogramming of Reactive Glial Cells into Functional Neurons after Brain Injury and in an Alzheimer’s Disease Model. Cell Stem Cell. 2014;14(2):188–202. doi: 10.1016/j.stem.2013.12.001Guo Z, Zhang L, Wu Z, Chen Y, Wang F, Chen G. In Vivo Direct Reprogramming of Reactive Glial Cells into Functional Neurons after Brain Injury and in an Alzheimer’s Disease Model. Cell Stem Cell. 2014;14(2):188–202. doi: 10.1016/j.stem.2013.12.001Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, Demircan T, et al. Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients. Cell Stem Cell. 2013;13(6):653–658. doi: 10.1016/j.stem.2013.11.002Schwank G, Koo BK, Sasselli V, Dekkers JF, Heo I, Demircan T, et al. Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients. Cell Stem Cell. 2013;13(6):653–658. doi: 10.1016/j.stem.2013.11.002Firth AL, Menon T, Parker GS, Qualls SJ, Lewis BM, Ke E, et al. Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs. Cell Reports. 2015;12(9):1385–1390. doi: 10.1016/j.celrep.2015.07.062Firth AL, Menon T, Parker GS, Qualls SJ, Lewis BM, Ke E, et al. Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs. Cell Reports. 2015;12(9):1385–1390. doi: 10.1016/j.celrep.2015.07.062Cho HM, Lee KH, Shen Y ming, Shin TJ, Ryu PD, Choi MC, et al. Transplantation of hMSCs Genome Edited with LEF1 Improves Cardio-Protective Effects in Myocardial Infarction. Molecular Therapy — Nucleic Acids. 2020;19:1186–1197. doi: 10.1016/j.omtn.2020.01.007Cho HM, Lee KH, Shen Y ming, Shin TJ, Ryu PD, Choi MC, et al. Transplantation of hMSCs Genome Edited with LEF1 Improves Cardio-Protective Effects in Myocardial Infarction. Molecular Therapy — Nucleic Acids. 2020;19:1186–1197. doi: 10.1016/j.omtn.2020.01.007О государственном регулировании в области генно-инженерной деятельности: федеральный закон от 12.07.1996 № 86-ФЗ; в ред. от 29.12.2022. Собрание законодательства РФ. 1996. № 28. Cт. 3348О государственном регулировании в области генно-инженерной деятельности: федеральный закон от 12.07.1996 № 86-ФЗ; в ред. от 29.12.2022. Собрание законодательства РФ. 1996. № 28. Cт. 3348Park S, Gwon Y, Khan SA, Jang KJ, Kim J. Engineering considerations of iPSC-based personalized medicine. Biomaterials Research. 2023;27(1):67. doi: 10.1186/s40824-023-00382-xPark S, Gwon Y, Khan SA, Jang KJ, Kim J. Engineering considerations of iPSC-based personalized medicine. Biomaterials Research. 2023;27(1):67. doi: 10.1186/s40824-023-00382-xKim C. Disease modeling and cell based therapy with iPSC: future therapeutic option with fast and safe application. Blood Research. 2014;49(1):7–14. doi: 10.5045/br.2014.49.1.7Kim C. Disease modeling and cell based therapy with iPSC: future therapeutic option with fast and safe application. Blood Research. 2014;49(1):7–14. doi: 10.5045/br.2014.49.1.7Gao X, Yang J, Tsang JCH, Ooi J, Wu D, Liu P. Reprogramming to Pluripotency Using Designer TALE Transcription Factors Targeting Enhancers. Stem Cell Reports. 2013;1(2):183– 197. doi: 10.1016/j.stemcr.2013.06.002Gao X, Yang J, Tsang JCH, Ooi J, Wu D, Liu P. Reprogramming to Pluripotency Using Designer TALE Transcription Factors Targeting Enhancers. Stem Cell Reports. 2013;1(2):183– 197. doi: 10.1016/j.stemcr.2013.06.002Weltner J, Balboa D, Katayama S, Bespalov M, Krjutškov K, Jouhilahti EM, et al. Human pluripotent reprogramming with CRISPR activators. Nature Communications. 2018;9(1):2643. doi: 10.1038/s41467-018-05067-xWeltner J, Balboa D, Katayama S, Bespalov M, Krjutškov K, Jouhilahti EM, et al. Human pluripotent reprogramming with CRISPR activators. Nature Communications. 2018;9(1):2643. doi: 10.1038/s41467-018-05067-xWeltner J, Trokovic R. Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators. Methods Mol Biol. 2021;2239:175–198. doi: 10.1007/978-1-0716-1084-8_12.Weltner J, Trokovic R. Reprogramming of Fibroblasts to Human iPSCs by CRISPR Activators. Methods Mol Biol. 2021;2239:175–198. doi: 10.1007/978-1-0716-1084-8_12.

The authors declare that there are no conflicts of interest present.