A recombineering pipeline to clone large and complex genes in Chlamydomonas.
Journal
The Plant cell
ISSN: 1532-298X
Titre abrégé: Plant Cell
Pays: England
ID NLM: 9208688
Informations de publication
Date de publication:
31 05 2021
31 05 2021
Historique:
received:
07
05
2020
accepted:
18
01
2021
pubmed:
17
3
2021
medline:
21
8
2021
entrez:
16
3
2021
Statut:
ppublish
Résumé
The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.
Identifiants
pubmed: 33723601
pii: 6126392
doi: 10.1093/plcell/koab024
pmc: PMC8633747
doi:
Substances chimiques
Epitopes
0
Recombinant Proteins
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1161-1181Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/R001014/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/S015337/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M011151/1
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BB/M011151/1A
Pays : United Kingdom
Informations de copyright
� American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.
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