Gene Targeting in the Red Alga <i>Cyanidioschyzon merolae</i>: Single- and Multi-Copy Insertion Using Authentic and Chimeric Selection Markers

<div><p>The unicellular red alga <i>Cyanidioschyzon merolae</i> is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, <i>URA_Cm-Gs</i>, has been available that complements the uracil-auxotrophic mutant M4. <i>URA_Cm-Gs</i>, a chimeric <i>URA5.3</i> gene of <i>C. merolae</i> and the related alga <i>Galdieria sulphuraria</i>, was originally designed to avoid gene conversion of the mutated <i>URA5.3</i> allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric <i>URA_Cm-Gs</i> selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic <i>URA5.3</i> gene (<i>URA_Cm-Cm</i>) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in <i>C. merolae.</i> Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.</p></div

Targeted insertion of <i>EGFP</i> by homologous recombination using <i>URA_Cm-Cm</i> and <i>URA_Cm-Gs</i> as selection markers.

<p>(<b>A</b>) Schematic diagram of the domain architectures of the two selection markers is shown on the top. Alignment of amino-acid sequences surrounding the OMP-decarboxylase domain of <i>C. merolae</i> and <i>G. sulphuraria</i> is shown in the bottom. The gray bar indicates the conserved OMP-decarboxylase domain. The red squares indicate amino-acid residues that play key roles in the enzymatic activity of OMP-decarboxylase <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073608#pone.0073608-Harris1" target="_blank">[20]</a>. (<b>B</b>) Schematic diagrams of targeted gene insertion by homologous recombination. The first line indicates the introduced DNA fragment, whereas the second line indicates the genomic structure of the parental strain M4. For efficient expression of <i>EGFP</i>, the 5′-UTR of the <i>CMO250C</i> gene and the 3′-UTR of the <i>β-tubulin</i> gene were utilized as a promoter and a putative polyadenylation signal sequence, respectively. The third and fourth lines indicate the predicted genomic structures in which a single copy is inserted by double-crossover homologous recombination in each case. The arrowheads indicate the positions of PCR primers used. (<b>C</b>) PCR analysis of CC and CG strains isolated independently, along with 10D (wild-type strain) and M4 (parental strain), to confirm homologous recombination events. Primers used were F1 (No. 25), R1 (No. 26), F2 (No. 27) and R2 (No. 28) shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073608#pone.0073608.s001" target="_blank">Table S1</a>. The predicted sizes of PCR products are as follows: F1/R1, 3.4 kb for CC and CG, no band for 10D and M4; F2/R2, 2.4 kb for CC and CG, no band for 10D and M4; F1/R2, 8.2 kb for CC and CG, 3.9 kb for 10D and M4.</p

Growth of and EGFP expression from the transformants.

<p>(<b>A</b>) Growth curves of the CC and CG strains in MA2 medium in the presence or absence of uracil. (<b>B</b>) Immunoblotting analysis against total lysates with anti-GFP. The arrowhead indicates the position of EGFP. A CBB-stained part of the gel is shown as a loading control. (<b>C</b>) Fluorescent images showing EGFP signals detected in the CC and CG strains. The CG strains emitted much higher levels of EGFP signals than the CC strains. Exposure times under blue-light excitation are 0.5 sec for 10 D, M4 and CC stains, and 0.2 sec for CG strains. The lower panels show typical G1 and M phase cells (CG1), indicating that the EGFP signals distributed throughout the cytosol. Note that the chloroplasts emitted red autofluorescence. Bars, 5 µm (upper panels) and 1 µm (lower panels). (<b>D</b>) Flow cytometry analysis of EGFP fluorescence. The broken line indicates the mode value calculated from the 10 D data, representing the sum of background and autofluorescence signals.</p