Targeted integration by homologous recombination enablesin situtagging and replacement of genes in the marine microeukaryoteDiplonema papillatum

Result code in IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60076658%3A12310%2F20%3A43901331" target="_blank" >RIV/60076658:12310/20:43901331 - isvavai.cz</a>

Alternative codes found

RIV/60077344:_____/20:00537342

Result on the web

<a href="https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.15130" target="_blank" >https://sfamjournals.onlinelibrary.wiley.com/doi/10.1111/1462-2920.15130</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1111/1462-2920.15130" target="_blank" >10.1111/1462-2920.15130</a>

Result language

angličtina

Original language name

Targeted integration by homologous recombination enablesin situtagging and replacement of genes in the marine microeukaryoteDiplonema papillatum

Original language description

Diplonemids are a group of highly diverse and abundant marine microeukaryotes that belong to the phylum Euglenozoa and form a sister clade to the well-studied, mostly parasitic kinetoplastids. Very little is known about the biology of diplonemids, as few species have been formally described and just one,Diplonema papillatum, has been studied to a decent extent at the molecular level. Following up on our previous results showing stable but random integration of delivered extraneous DNA, we demonstrate here homologous recombination inD. papillatum. Targeting various constructs to the intended position in the nuclear genome was successful when 5 &apos; and 3 &apos; homologous regions longer than 1 kbp were used, achieving N-terminal tagging with mCherry and gene replacement of alpha- and beta-tubulins. For more convenient genetic manipulation, we designed a modular plasmid, pDP002, which bears a protein-A tag and used it to generate and express a C-terminally tagged mitoribosomal protein. Lastly, we developed an improved transformation protocol for broader applicability across laboratories. Our robust methodology allows the replacement, integration as well as endogenous tagging ofD. papillatumgenes, thus opening the door to functional studies in this species and establishing a basic toolkit for reverse genetics of diplonemids in general.

Czech name

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Czech description

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Type

J_imp - Article in a specialist periodical, which is included in the Web of Science database

CEP classification

—

OECD FORD branch

10606 - Microbiology

Project

Result was created during the realization of more than one project. More information in the Projects tab.

Continuities

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Publication year

2020

Confidentiality

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Name of the periodical

Environmental Microbiology

ISSN

1462-2912

e-ISSN

—

Volume of the periodical

22

Issue of the periodical within the volume

9

Country of publishing house

GB - UNITED KINGDOM

Number of pages

11

Pages from-to

3660-3670

UT code for WoS article

000552503400001

EID of the result in the Scopus database

2-s2.0-85088447871