vavilovВавиловский журнал генетики и селекцииVavilov Journal of Genetics and Breeding2500-3259Institute of Cytology and Genetics of Siberian Branch of the RAS10.18699/VJ19.483vavilov-1937Research ArticleРЕГУЛЯЦИЯ ГЕНОВ И ГЕНОМОВGENOME AND GENE REGULATIONСоздание штрихкодированных плазмидных библиотек для множественного одновременного анализа эффекта положения генаGeneration of barcoded plasmid libraries for massively parallel analysis of chromatin position effectshttps://orcid.org/0000-0001-7461-0977ЛебедевМ. О.LebedevM. O.

Новосибирск.

Novosibirsk.

https://orcid.org/0000-0003-0469-0371ЯриничЛ. А.YarinichL. A.

Новосибирск.

Novosibirsk.

https://orcid.org/0000-0003-3309-6363ИванкинА. В.IvankinA. V.

Новосибирск.

Novosibirsk.

https://orcid.org/0000-0001-6959-0641ПиндюринА. В.PindyurinA. V.

Новосибирск.

Novosibirsk.

Институт молекулярной и клеточной биологии Сибирского отделения Российской академии наук; Новосибирский национальный исследовательский государственный университет.РоссияInstitute of Molecular and Cellular Biology, SB RAS; Novosibirsk State University.Russian FederationИнститут молекулярной и клеточной биологии Сибирского отделения Российской академии наук.РоссияInstitute of Molecular and Cellular Biology, SB RAS.Russian Federation201930032019232203211Copyright © Лебедев М.О., Яринич Л.А., Иванкин А.В., Пиндюрин А.В., 20192019Лебедев М.О., Яринич Л.А., Иванкин А.В., Пиндюрин А.В.Lebedev M.O., Yarinich L.A., Ivankin A.V., Pindyurin A.V.This work is licensed under a Creative Commons Attribution 4.0 License.https://vavilov.elpub.ru/jour/article/view/1937

Открытие явления мозаичного эффекта положения гена и последующий тщательный анализ его молекулярных механизмов привели к пониманию того, что локальный состав хроматина оказывает существенное влияние на активность генов. Разработанный недавно метод Thousands of Reporters Integrated in Parallel (TRIP) основан на использовании штрихкодированных генов-репортеров и позволяет выполнять высокопроизводительный анализ эффекта положения гена в масштабе всего генома. В настоящей работе мы описываем конструирование и проверку качества штрихкодированных плазмидных библиотек высокой сложности, которые предполагается использовать для высокопроизводительного анализа эффекта положения гена в клетках дрозофилы. Во-первых, рассмотрены наиболее важные параметры, которые следует учитывать при создании штрихкодированных плазмидных библиотек, и предложен простой метод оценки сложности вырожденного фрагмента (штрихкода) в синтетических олигонуклеотидах с использованием ПЦР-амплификации и последующего секвенирования по методу Сэнгера. Далее проведено сравнение традиционного метода клонирования посредством рестрикции – лигирования с подходом сборки по методу Гибсона для клонирования штрихкодов в один и тот же плазмидный вектор. Кроме того, описаны оптимизированные параметры для создания штрихкодированных плазмидных библиотек, такие как соотношение вектор : вставка в реакции сборки методом Гибсона и напряжение, используемое для электропорации бактериальных клеток продуктами лигирования. Сравниваются также различные подходы для проверки качества штрихкодированных плазмидных библиотек. Наконец, кратко описаны альтернативные подходы, которые можно использовать для создания таких библиотек. Важно отметить, что все улучшения и модификации методов, приведенные в данной работе, могут быть применены к широкому кругу экспериментов, в которых используются штрихкодированные плазмидные библиотеки.

The discovery of the position effect variegation phenomenon and the subsequent comprehensive analysis of its molecular mechanisms led to understanding that the local chromatin composition has a dramatic effect on gene activity. To study this effect in a high-throughput mode and at the genome-wide level, the Thousands of Reporters Integrated in Parallel (TRIP) approach based on the usage of barcoded reporter gene constructs was recently developed. Here we describe the construction and quality checks of high-diversity barcoded plasmid libraries supposed to be used for high-throughput analysis of chromatin position effects in Drosophila cells. First, we highlight the critical parameters that should be considered in the generation of barcoded plasmid libraries and introduce a simple method to assess the diversity of random sequences (barcodes) of synthetic oligonucleotides using PCR amplification followed by Sanger sequencing. Second, we compare the conventional restriction-ligation method with the Gibson assembly approach for cloning barcodes into the same plasmid vector. Third, we provide optimized parameters for the construction of barcoded plasmid libraries, such as the vector : insert ratio in the Gibson assembly reaction and the voltage used for electroporation of bacterial cells with ligation products. We also compare different approaches to check the quality of barcoded plasmid libraries. Finally, we briefly describe alternative approaches that can be used for the generation of such libraries. Importantly, all improvements and modifications of the techniques described here can be applied to a wide range of experiments involving barcoded plasmid libraries.

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