Targeted gene silencing by RNA interference in Chlamydomonas.

Eun Jeong Kim, Heriberto Cerutti

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms and plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. Double-stranded RNA (dsRNA) triggered posttranscriptional gene silencing, termed RNA interference (RNAi), is also becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of long dsRNAs or duplex small RNAs from genome-integrated transgenes, has been achieved in multiple organisms, including the green alga Chlamydomonas reinhardtii. However, the level of gene repression is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. Moreover, unintended transcripts partly complementary to a trigger dsRNA can also be silenced, making difficult the interpretation of observed phenotypes. To obviate some of these problems we have developed a tandem inverted repeat system that consistently induces cosilencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta-subunit) and any other (nonessential) gene of interest. In addition, to circumvent off-target effects, for each tested gene, RNAi lines are generated with at least two transgenes, homologous to distinct and nonoverlapping sequences of the target transcript. A common phenotype among these independent RNAi strains is expected to result from suppression of expression of the gene of interest. We demonstrate this approach for the characterization of a gene of unknown function in Chlamydomonas, encoding a predicted exoribonuclease with weak similarity to 3'hExo/ERI-1. 2009 Elsevier Inc. All rights reserved.

Original languageEnglish (US)
Pages (from-to)99-110
Number of pages12
JournalMethods in cell biology
Volume93
DOIs
StatePublished - 2009

Fingerprint

Chlamydomonas
Gene Silencing
RNA Interference
Transgenes
Genes
Double-Stranded RNA
Phenotype
Exoribonucleases
Tryptophan Synthase
Inverted Repeat Sequences
RNA
Reverse Genetics
Chlamydomonas reinhardtii
Chlorophyta
Eukaryota
Parasites
Genome
Gene Expression

ASJC Scopus subject areas

  • Cell Biology

Cite this

Targeted gene silencing by RNA interference in Chlamydomonas. / Kim, Eun Jeong; Cerutti, Heriberto.

In: Methods in cell biology, Vol. 93, 2009, p. 99-110.

Research output: Contribution to journalArticle

@article{d5d8fab3a2234abbadf9845fdf7133fa,
title = "Targeted gene silencing by RNA interference in Chlamydomonas.",
abstract = "Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms and plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. Double-stranded RNA (dsRNA) triggered posttranscriptional gene silencing, termed RNA interference (RNAi), is also becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of long dsRNAs or duplex small RNAs from genome-integrated transgenes, has been achieved in multiple organisms, including the green alga Chlamydomonas reinhardtii. However, the level of gene repression is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. Moreover, unintended transcripts partly complementary to a trigger dsRNA can also be silenced, making difficult the interpretation of observed phenotypes. To obviate some of these problems we have developed a tandem inverted repeat system that consistently induces cosilencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta-subunit) and any other (nonessential) gene of interest. In addition, to circumvent off-target effects, for each tested gene, RNAi lines are generated with at least two transgenes, homologous to distinct and nonoverlapping sequences of the target transcript. A common phenotype among these independent RNAi strains is expected to result from suppression of expression of the gene of interest. We demonstrate this approach for the characterization of a gene of unknown function in Chlamydomonas, encoding a predicted exoribonuclease with weak similarity to 3'hExo/ERI-1. 2009 Elsevier Inc. All rights reserved.",
author = "Kim, {Eun Jeong} and Heriberto Cerutti",
year = "2009",
doi = "10.1016/S0091-679X(08)93005-3",
language = "English (US)",
volume = "93",
pages = "99--110",
journal = "Methods in Cell Biology",
issn = "0091-679X",
publisher = "Academic Press Inc.",

}

TY - JOUR

T1 - Targeted gene silencing by RNA interference in Chlamydomonas.

AU - Kim, Eun Jeong

AU - Cerutti, Heriberto

PY - 2009

Y1 - 2009

N2 - Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms and plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. Double-stranded RNA (dsRNA) triggered posttranscriptional gene silencing, termed RNA interference (RNAi), is also becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of long dsRNAs or duplex small RNAs from genome-integrated transgenes, has been achieved in multiple organisms, including the green alga Chlamydomonas reinhardtii. However, the level of gene repression is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. Moreover, unintended transcripts partly complementary to a trigger dsRNA can also be silenced, making difficult the interpretation of observed phenotypes. To obviate some of these problems we have developed a tandem inverted repeat system that consistently induces cosilencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta-subunit) and any other (nonessential) gene of interest. In addition, to circumvent off-target effects, for each tested gene, RNAi lines are generated with at least two transgenes, homologous to distinct and nonoverlapping sequences of the target transcript. A common phenotype among these independent RNAi strains is expected to result from suppression of expression of the gene of interest. We demonstrate this approach for the characterization of a gene of unknown function in Chlamydomonas, encoding a predicted exoribonuclease with weak similarity to 3'hExo/ERI-1. 2009 Elsevier Inc. All rights reserved.

AB - Small RNA-guided gene silencing is an evolutionarily conserved process that operates by a variety of molecular mechanisms and plays an essential role in developmental pathways and defense responses against genomic parasites in eukaryotes. Double-stranded RNA (dsRNA) triggered posttranscriptional gene silencing, termed RNA interference (RNAi), is also becoming a powerful tool for reverse genetics studies. Stable RNAi, induced by the expression of long dsRNAs or duplex small RNAs from genome-integrated transgenes, has been achieved in multiple organisms, including the green alga Chlamydomonas reinhardtii. However, the level of gene repression is often quite variable, depending on the type of construct, transgene copy number, site of integration, and target gene. Moreover, unintended transcripts partly complementary to a trigger dsRNA can also be silenced, making difficult the interpretation of observed phenotypes. To obviate some of these problems we have developed a tandem inverted repeat system that consistently induces cosilencing of a gene with a selectable RNAi-induced phenotype (encoding tryptophan synthase beta-subunit) and any other (nonessential) gene of interest. In addition, to circumvent off-target effects, for each tested gene, RNAi lines are generated with at least two transgenes, homologous to distinct and nonoverlapping sequences of the target transcript. A common phenotype among these independent RNAi strains is expected to result from suppression of expression of the gene of interest. We demonstrate this approach for the characterization of a gene of unknown function in Chlamydomonas, encoding a predicted exoribonuclease with weak similarity to 3'hExo/ERI-1. 2009 Elsevier Inc. All rights reserved.

UR - http://www.scopus.com/inward/record.url?scp=77954676702&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77954676702&partnerID=8YFLogxK

U2 - 10.1016/S0091-679X(08)93005-3

DO - 10.1016/S0091-679X(08)93005-3

M3 - Article

C2 - 20409813

AN - SCOPUS:77954676702

VL - 93

SP - 99

EP - 110

JO - Methods in Cell Biology

JF - Methods in Cell Biology

SN - 0091-679X

ER -