wdr92 was generated by transformation with electroporation of DNA fragments, which harbor expression cassette of paromomycin resistant gene AphVIII (Sizova et al., 2001 (link)). The flanking sequences of the DNA insert were identified by restriction enzyme site-directed PCR (RESDA-PCR) followed by sequencing (Gonzalez-Ballester et al., 2005 (link)). For gene complementation experiments, WDR92, MOT48, TWI1 genes with an ~1.5 kb fragment upstream of the start codon were cloned respectively into the modified vector pHyg3 harboring a hygromycin B resistance gene with a YFP or 3× HA tag at the 3′ end (Berthold et al., 2002 (link); Meng et al., 2014 (link)). For pf13 complementation, a full-length cDNA for PF13, driven by PsaD promoter and tagged with a 3× HA tag at the 3′ end (Fischer and Rochaix, 2001 (link)), was also constructed into the modified vector pHyg3. The final constructs were linearized with ScaI and transformed into the respective mutants using electroporation (Liang and Pan, 2013 (link)). All the cDNAs used in this study were cloned from a Chlamydomonas cDNA library (TaKaRa). All the constructs were verified by sequencing.
Chlamydomonas cdna library
Manufactured by Takara Bio
Sourced in China
The Chlamydomonas cDNA library is a collection of complementary DNA (cDNA) clones derived from the model organism Chlamydomonas reinhardtii. The library serves as a valuable resource for researchers studying the molecular biology, genetics, and gene expression of this green algae species.
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3 protocols using chlamydomonas cdna library
1
Generation of Chlamydomonas Mutant Complementation Constructs
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wdr92 was generated by transformation with electroporation of DNA fragments, which harbor expression cassette of paromomycin resistant gene AphVIII (Sizova et al., 2001 (link)). The flanking sequences of the DNA insert were identified by restriction enzyme site-directed PCR (RESDA-PCR) followed by sequencing (Gonzalez-Ballester et al., 2005 (link)). For gene complementation experiments, WDR92, MOT48, TWI1 genes with an ~1.5 kb fragment upstream of the start codon were cloned respectively into the modified vector pHyg3 harboring a hygromycin B resistance gene with a YFP or 3× HA tag at the 3′ end (Berthold et al., 2002 (link); Meng et al., 2014 (link)). For pf13 complementation, a full-length cDNA for PF13, driven by PsaD promoter and tagged with a 3× HA tag at the 3′ end (Fischer and Rochaix, 2001 (link)), was also constructed into the modified vector pHyg3. The final constructs were linearized with ScaI and transformed into the respective mutants using electroporation (Liang and Pan, 2013 (link)). All the cDNAs used in this study were cloned from a Chlamydomonas cDNA library (TaKaRa). All the constructs were verified by sequencing.
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wdr92 was generated by transformation with electroporation of DNA fragments, which harbor expression cassette of paromomycin resistant gene AphVIII (Sizova et al., 2001 (link)). The flanking sequences of the DNA insert were identified by restriction enzyme site-directed PCR (RESDA-PCR) followed by sequencing (Gonzalez-Ballester et al., 2005 (link)). For gene complementation experiments, WDR92, MOT48, TWI1 genes with an ~1.5 kb fragment upstream of the start codon were cloned respectively into the modified vector pHyg3 harboring a hygromycin B resistance gene with a YFP or 3× HA tag at the 3′ end (Berthold et al., 2002 (link); Meng et al., 2014 (link)). For pf13 complementation, a full-length cDNA for PF13, driven by PsaD promoter and tagged with a 3× HA tag at the 3′ end (Fischer and Rochaix, 2001 (link)), was also constructed into the modified vector pHyg3. The final constructs were linearized with ScaI and transformed into the respective mutants using electroporation (Liang and Pan, 2013 (link)). All the cDNAs used in this study were cloned from a Chlamydomonas cDNA library (TaKaRa). All the constructs were verified by sequencing.
2
Chimeric Kinesin Motor Protein Engineering
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Full-length cDNAs of KIF3A and KIFAP3 were gifts of Dr. Jiahuai Han (Xiamen University, China). Full-length cDNA of KIF3B was synthesized (WuXi Qinglan Biotech). FLA10, FLA8 and KAP cDNAs were cloned from a Chlamydomonas cDNA library (Takara). For chimeric kinesins, the motor domain of FLA10 was replaced with that of FLA8 or KIF3A to generate FLA8’ or KIF3A’ as specified in the text, respectively. The constructs for chimeric kinesins FLA10’ and KIF3B’ were similarly generated. The cDNAs with tags as indicated in the text were cloned in the pOCC vectors, respectively, by conventional molecular techniques.
3
Insertional Mutagenesis in Chlamydomonas
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Previously published protocols were used to generate insertional mutants by transformation of Chlamydomonas wild type cells with a ~ 2 kb fragment containing the paromomycin resistance gene AphVIII[53 (link)]. The disrupted genes were identified by cloning the flanking genomic sequences using RESDA PCR followed by sequencing [54 (link), 55 (link)]. The DNA fragment with about 250 nt being deleted from 5’ end has replaced the nucleotides 215–221 of the IFT43 gene. The insertion information of other IFT-A mutants is provided in the supplemental data. The IFT43 gene with an approximately 1.5 kb fragment upstream of the start codon was cloned by PCR and verified by sequencing. To rescue the mutant phenotype of ift43, the IFT43 gene was tagged with 3×HA or YFP followed by a RUBISCO terminator. Deletion mutants of the IFT43 gene were constructed based on the wild type IFT43 gene construct. These constructs were cloned into a modified vector pHyg3 harboring a hygromycine B resistance gene[56 (link)]. The final constructs were linearized with ScaI and transformed into the ift43 mutant using electroporation[57 (link)]. The cDNAs for the IFT-A genes were cloned from a Chlamydomonas cDNA library (Takara, Dalian, China).
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