Building a multipurpose insertional mutant library for forward and reverse genetics in Chlamydomonas

Plant Methods
Xi ChengKaiyao Huang

Abstract

The unicellular green alga, Chlamydomonas reinhardtii, is a classic model for studying flagella and biofuel. However, precise gene editing, such as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas9) system, is not widely used in this organism. Screening of random insertional mutant libraries by polymerase chain reaction provides an alternate strategy to obtain null mutants of individual gene. But building, screening, and maintaining such a library was time-consuming and expensive. By selecting a suitable parental strain, keeping individual mutants using the agar plate, and designing an insertion cassette-specific primer for library screening, we successfully generated and maintained ~150,000 insertional mutants of Chlamydomonas, which was used for both reverse and forward genetics analysis. We obtained 26 individual mutants corresponding to 20 genes and identified 967 motility-defect mutants including 10 mutants with defective accumulation of intraflagellar transport complex at the basal body. We also obtained 929 mutants defective in oil droplet assembly after nitrogen deprivation. Furthermore, a new insertion cassette with splicing donor sequences at both ends was also cons...Continue Reading

References

Feb 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·K L Kindle
May 1, 1990·Molecular & General Genetics : MGG·J A Schloss
Mar 1, 1985·The Journal of Cell Biology·P GreenspanS D Fowler
Jan 1, 1968·Molecular & General Genetics : MGG·R Loppes
Apr 24, 1996·Molecular & General Genetics : MGG·D R StevensS Purton
May 5, 2001·Annual Review of Plant Physiology and Plant Molecular Biology·Elizabeth H Harris
Aug 1, 1959·Canadian Journal of Biochemistry and Physiology·E G BLIGH, W J DYER
Jan 25, 2005·Plant Physiology·Linda A RymarquisDavid B Stern
Jan 25, 2005·Plant Physiology·David González-BallesterEmilio Fernández
Apr 21, 2005·Analytical Biochemistry·David González-BallesterEmilio Fernández
Jul 7, 2005·The Journal of Cell Biology·Gregory J PazourGeorge B Witman
Nov 26, 2005·Current Genetics·Michael Schroda
Jun 1, 1965·Proceedings of the National Academy of Sciences of the United States of America·D C Fork, W Urbach
Oct 13, 2007·Science·Sabeeha S MerchantArthur R Grossman
Nov 7, 2007·The Journal of Cell Biology·Kaiyao HuangJoel L Rosenbaum
Nov 28, 2008·The Plant Journal : for Cell and Molecular Biology·Juliane NeupertRalph Bock
Dec 5, 2008·The Plant Journal : for Cell and Molecular Biology·Attila MolnarDavid Baulcombe
Feb 27, 2009·Protoplasma·Muqing CaoJunmin Pan
Dec 8, 2009·Current Opinion in Cell Biology·Cosima T Baldari, Joel Rosenbaum
Jul 29, 2011·Plant Methods·David Gonzalez-BallesterArthur R Grossman
Jan 3, 2012·Current Opinion in Biotechnology·Sabeeha S MerchantJaruswan Warakanont
Mar 3, 2012·G3 : Genes - Genomes - Genetics·Susan K DutcherBrian L Lewis
Nov 10, 2012·The Plant Journal : for Cell and Molecular Biology·Irina SizovaPeter Hegemann
Mar 19, 2013·Methods in Enzymology·Huan Long, Kaiyao Huang
Mar 26, 2013·The Plant Journal : for Cell and Molecular Biology·Beth A RasalaStephen P Mayfield
Jul 1, 2014·The Plant Journal : for Cell and Molecular Biology·Jinlu HuKaiyao Huang
Oct 1, 2014·The Plant Journal : for Cell and Molecular Biology·Mia TerashimaMartin C Jonikas
Feb 3, 2015·The Plant Journal : for Cell and Molecular Biology·Melissa A ScrantonStephen P Mayfield
Feb 19, 2015·The Plant Journal : for Cell and Molecular Biology·Ursula Goodenough
Apr 25, 2015·Plant Methods·Sergio A Garcia-Echauri, Guy A Cardineau
Sep 25, 2015·The Plant Journal : for Cell and Molecular Biology·Rouhollah BarahimipourRalph Bock

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Citations

May 23, 2019·Molecular Biology of the Cell·Erin E DymekElizabeth F Smith
Mar 7, 2020·Clinical Journal of the American Society of Nephrology : CJASN·Elisa Molinari, John A Sayer
Oct 6, 2020·Frontiers in Bioengineering and Biotechnology·Gulshan KumarTilak Raj Sharma
Apr 21, 2021·Proceedings of the National Academy of Sciences of the United States of America·Lijuan ZhaoKaiyao Huang
Sep 2, 2021·Journal of Experimental Botany·Yong Zou, Peter V Bozhkov

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Methods Mentioned

BETA
PCR
3′-phosphotransferase
confocal microscopy
fluorescence microscopy
glycosylation
electrophoresis
Assay

Software Mentioned

RESDA
NIS
Primer Premier
Elements BR
SDS
Quantity One
PCR

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