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Pitfalls of using sequence databases for heterologous expression studies - a technical review

  • Synthesis of DNA fragments based on gene sequences available in public resources has become an efficient and affordable method that gradually replaced traditional cloning efforts such as PCR cloning from cDNA. However, database entries based on genome sequencing results are prone to errors which can lead to false sequence information and, ultimately, errors in functional characterization of proteins such as ion channels and transporters in heterologous expression systems. We have identified five common problems that repeatedly appear in public resources: 1) Not every gene has yet been annotated; 2) Not all gene annotations are necessarily correct; 3) Transcripts may contain automated corrections; 4) There are mismatches between gene, mRNA, and protein sequences; and 5) Splicing patterns often lack experimental validation. This technical review highlights and provides a strategy to bypass these issues in order to avoid critical mistakes that could impact future studies of any gene/protein of interest in heterologous expression systems. Abstract figure legend Projects involving heterologous gene expression are often characterised by similar steps. Initially, database research (A) is necessary to retrieve information of full of partial sequences of a gene of interest. A multitude of genome assemblies are annotated and deposited in public databases or that are available for refined search options using individual sequence information. The search results need to be scrutinised and compared to already available information (B). Once the sequence has been determined, DNA synthesis (C) by PCR or commercial synthesis are necessary for further cloning procedures (D). Eventually, the DNA needs to be transfected (E) and expressed in, e.g., eukaryotic cells (F). Finally, the expression of the gene of interest needs to be documented and its function analysed (G). This article is protected by copyright. All rights reserved.

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Document Type:Article
Author:Stephan Maxeiner, Gabriela Krasteva-Christ, Mike Althaus
Parent Title (English):The Journal of Physiology
First Page:1611
Last Page:1623
Place of publication:Hoboken, NJ
Publishing Institution:Hochschule Bonn-Rhein-Sieg
Date of first publication:2023/02/10
Copyright:© 2023 The Authors. The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.
Funding:S.M. received funding from the Medical School of SaarlandUniversity: HOMFOR2017-19. G.K.C. is supported by theGerman Research Foundation (DFG): KR4338/1-2 and SFBTRR152 P22. M.A. receives funding from the Ministry ofCulture and Science of the State of North Rhine-Westphalia: FKZ 005-2101-0144 and FKZ 005-2211-0043, and DFG: INST19446/3-1.
Keyword:gene expression; genomic data; rodents; sequencing
Departments, institutes and facilities:Fachbereich Angewandte Naturwissenschaften
Institut für funktionale Gen-Analytik (IFGA)
Dewey Decimal Classification (DDC):6 Technik, Medizin, angewandte Wissenschaften / 61 Medizin und Gesundheit / 612 Humanphysiologie
Entry in this database:2023/03/23
Licence (German):License LogoCreative Commons - CC BY-NC - Namensnennung - Nicht kommerziell 4.0 International