Skip to main content

Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii.

Publication ,  Journal Article
Onishi, M; Pringle, JR
Published in: G3 (Bethesda, Md.)
December 2016

The unicellular green alga Chlamydomonas reinhardtii is a model organism that provides an opportunity to understand the evolution and functional biology of the lineage that includes the land plants, as well as aspects of the fundamental core biology conserved throughout the eukaryotic phylogeny. Although many tools are available to facilitate genetic, molecular biological, biochemical, and cell biological studies in Chlamydomonas, expression of unselected transgenes of interest (GOIs) has been challenging. In most methods used previously, the GOI and a selectable marker are expressed from two separate mRNAs, so that their concomitant expression is not guaranteed. In this study, we developed constructs that allow expression of an upstream GOI and downstream selectable marker from a single bicistronic mRNA. Although this approach in other systems has typically required a translation-enhancing element such as an internal ribosome entry site for the downstream marker, we found that a short stretch of unstructured junction sequence was sufficient to obtain adequate expression of the downstream gene, presumably through post-termination reinitiation. With this system, we obtained robust expression of both endogenous and heterologous GOIs, including fluorescent proteins and tagged fusion proteins, in the vast majority of transformants, thus eliminating the need for tedious secondary screening for GOI-expressing transformants. This improved efficiency should greatly facilitate a variety of genetic and cell-biological studies in Chlamydomonas and also enable new applications such as expression-based screens and large-scale production of foreign proteins.

Duke Scholars

Altmetric Attention Stats
Dimensions Citation Stats

Published In

G3 (Bethesda, Md.)

DOI

EISSN

2160-1836

ISSN

2160-1836

Publication Date

December 2016

Volume

6

Issue

12

Start / End Page

4115 / 4125

Related Subject Headings

  • Transgenes
  • Transformation, Genetic
  • RNA, Messenger
  • Promoter Regions, Genetic
  • Genetic Vectors
  • Genetic Markers
  • Gene Order
  • Gene Expression
  • Chlamydomonas reinhardtii
  • 4905 Statistics
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Onishi, M., & Pringle, J. R. (2016). Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda, Md.), 6(12), 4115–4125. https://doi.org/10.1534/g3.116.033035
Onishi, Masayuki, and John R. Pringle. “Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii.G3 (Bethesda, Md.) 6, no. 12 (December 2016): 4115–25. https://doi.org/10.1534/g3.116.033035.
Onishi M, Pringle JR. Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda, Md). 2016 Dec;6(12):4115–25.
Onishi, Masayuki, and John R. Pringle. “Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii.G3 (Bethesda, Md.), vol. 6, no. 12, Dec. 2016, pp. 4115–25. Epmc, doi:10.1534/g3.116.033035.
Onishi M, Pringle JR. Robust Transgene Expression from Bicistronic mRNA in the Green Alga Chlamydomonas reinhardtii. G3 (Bethesda, Md). 2016 Dec;6(12):4115–4125.

Published In

G3 (Bethesda, Md.)

DOI

EISSN

2160-1836

ISSN

2160-1836

Publication Date

December 2016

Volume

6

Issue

12

Start / End Page

4115 / 4125

Related Subject Headings

  • Transgenes
  • Transformation, Genetic
  • RNA, Messenger
  • Promoter Regions, Genetic
  • Genetic Vectors
  • Genetic Markers
  • Gene Order
  • Gene Expression
  • Chlamydomonas reinhardtii
  • 4905 Statistics