vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ21.014vavilov-2924Research ArticleБИОТЕХНОЛОГИЯBIOTECHNOLOGYТрансгенная клеточная линия с индуцируемой транскрипцией для исследования механизмов экспансии (CGG)n повторовA transgenic cell line with inducible transcription for studying (CGG)n repeat expansion mechanismshttps://orcid.org/0000-0002-2227-8500ГрищенкоИ. В.GrishchenkoI. V.

р.п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk region

https://orcid.org/0000-0002-1277-4113ТулуповА. А.TulupovA. A.

Новосибирск

Novosibirsk

РымареваЮ. М.RymarevaY. M.

Новосибирск

Novosibirsk

https://orcid.org/0000-0003-4325-4062ПетровскийЕ. Д.PetrovskiyE. D.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-5332-2607СавеловА. А.SavelovA. A.

Новосибирск

Novosibirsk

https://orcid.org/0000-0002-0095-8994КоростышевскаяА. М.KorostyshevskayaA. M.

Новосибирск

Novosibirsk

МаксимоваЮ. В.MaksimovaY. V.

Новосибирск

Novosibirsk

ШоринаА. Р.ShorinaA. R.

Новосибирск

Novosibirsk

https://orcid.org/0000-0001-8529-9176ШитикЕ. М.ShitikE. M.

р.п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk region

https://orcid.org/0000-0002-8940-9173ЮдкинД. В.YudkinD. V.

р.п. Кольцово, Новосибирская область

Koltsovo, Novosibirsk region

yudkin_dv@vector.nsc.ruГосударственный научный центр вирусологии и биотехнологии «Вектор» РоспотребнадзораРоссияState Research Center of Virology and Biotechnology “Vector”, RospotrebnadzorRussian FederationИнститут «Международный томографический центр» Сибирского отделения Российской академии наук; Новосибирский национальный исследовательский государственный университетРоссияInternational Tomography Center of Siberian Branch of the Russian Academy of Sciences; Novosibirsk State UniversityRussian FederationИнститут «Международный томографический центр» Сибирского отделения Российской академии наукРоссияInternational Tomography Center of Siberian Branch of the Russian Academy of SciencesRussian FederationНовосибирский государственный медицинский университет; Городская клиническая больница № 1РоссияNovosibirsk State Medical University; Novosibirsk City Clinical Hospital No. 1Russian FederationГородская клиническая больница № 1РоссияNovosibirsk City Clinical Hospital No. 1Russian Federation202115032021251117124Copyright © Грищенко И.В., Тулупов А.А., Рымарева Ю.М., Петровский Е.Д., Савелов А.А., Коростышевская А.М., Максимова Ю.В., Шорина А.Р., Шитик Е.М., Юдкин Д.В., 20212021Грищенко И.В., Тулупов А.А., Рымарева Ю.М., Петровский Е.Д., Савелов А.А., Коростышевская А.М., Максимова Ю.В., Шорина А.Р., Шитик Е.М., Юдкин Д.В.Grishchenko I.V., Tulupov A.A., Rymareva Y.M., Petrovskiy E.D., Savelov A.A., Korostyshevskaya A.M., Maksimova Y.V., Shorina A.R., Shitik E.M., Yudkin D.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/2924

Существует ряд наследственных заболеваний человека, причиной которых является экспансия тандемных повторов. К ним относятся миотоническая дистрофия первого типа, болезнь Хантингтона, заболевания, ассоциированные с ломкой Х-­хромосомой. Синдром ломкой Х­-хромосомы – наиболее распространенная причина наследственной умственной отсталости у человека. На сегодняшний день причины развития экспансии остаются неисследованными. Важная особенность протяженных повторов – их способность формировать альтернативные вторичные структуры ДНК. Существуют гипотезы, объясняющие природу нестабильности повторов, однако все они предполагают возникновение устойчивых вторичных структур ДНК на различных этапах клеточного цикла. Источником нестабильности считаются нарушения в различных процессах метаболизма ДНК (репликация, репарация и рекомбинация), вызванные образованием вторичных структур. Однако ни одна из гипотез до конца не подтверждена и, видимо, не является единственно верной. Вероятно, в различных типах клеток и на определенных стадиях клеточного цикла источником нестабильности выступает множество процессов. В настоящей работе мы предлагаем экспериментальную систему для изучения вклада транскрипции и ассоциированной с ней репарации в нестабильность повтора (CGG)n, поскольку это наименее изученный механизм возникновения нестабильности. Однако предложенные модели могут учитывать вклад и других процессов метаболизма ДНК, например репликации, что делает полученные системы универсальными и применимыми в разных исследованиях. Нами были созданы трансгенные клеточные линии, несущие повтор нормальной и премутантной длины под тетрациклин­индуцируемым промотором. Один тип линий содержит плазмиду с экзогенным повтором, интегрированным в геном посредством транспозона Sleeping Beauty, в другой клеточной линии вектор поддерживается в виде эписомы благодаря ориджину репликации SV40. Такие трансгенные клеточные линии могут служить экспериментальной системой для поиска причин нестабильности и создания терапевтических средств. Кроме того, был разработан критерий для оценки нестабильности экзогенного (CGG)n повтора в геноме трансгенных клеточных линий, расчет которого не зависит от эффективности синтеза протяженных повторов.

There are more than 30 inherited human disorders connected with repeat expansion (myotonic dystrophy type I, Huntington’s disease, Fragile X syndrome). Fragile X syndrome is the most common reason for inherited intellectual disability in the human population. The ways of the expansion development remain unclear. An important feature of expanded repeats is the ability to form stable alternative DNA secondary structures. There are hypotheses about the nature of repeat instability. It is proposed that these DNA secondary structures can block various stages of DNA metabolism processes, such as replication, repair and recombination and it is considered as the source of repeat instability. However, none of the hypotheses is fully conf irmed or is the only valid one. Here, an experimental system for studying (CGG)n repeat expansion associated with transcription and TCR­-NER is proposed. It is noteworthy that the aberrations of transcription are a poorly studied mechanism of (CGG)n instability. However, the proposed systems take into account the contribution of other processes of DNA metabolism and, therefore, the developed systems are universal and applicable for various studies. Transgenic cell lines carrying a repeat of normal or premutant length under the control of an inducible promoter were established and a method for repeat instability quantif ication was developed. One type of the cell lines contains an exogenous repeat integrated into the genome by the Sleeping Beauty transposon; in another cell line, the vector is maintained as an episome due to the SV40 origin of replication. These experimental systems can serve for f inding the causes of instability and the development of therapeutic agents. In addition, a criterion was developed for the quantif ication of exogenous (CGG)n repeat instability in the transgenic cell lines’ genome.

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