TY  - JOUR
U1  - Zeitschriftenartikel, wissenschaftlich - begutachtet (reviewed)
A1  - Maxeiner, Stephan
A1  - Krasteva-Christ, Gabriela
A1  - Althaus, Mike
T1  - Pitfalls of using sequence databases for heterologous expression studies - a technical review
JF  - The Journal of Physiology
N2  - 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.
KW  - gene expression
KW  - genomic data
KW  - rodents
KW  - sequencing
UN  - https://nbn-resolving.org/urn:nbn:de:hbz:1044-opus-67145
SN  - 0022-3751
SS  - 0022-3751
U6  - https://doi.org/10.1113/JP284066
DO  - https://doi.org/10.1113/JP284066
PM  - 36762618
VL  - 601
IS  - 9
SP  - 1611
EP  - 1623
PB  - Wiley-Blackwell
CY  - Hoboken, NJ
ER  -