Reverse genetics in Chlamydomonas: a platform for isolating insertional mutants

Abstract

A method was developed to identify insertional mutants of Chlamydomonas reinhardtii disrupted for selected target genes. The approach relies on the generation of thousands of transformants followed by PCR-based screenings that allow for identification of strains harboring the introduced marker gene within specific genes of interest. Our results highlight the strengths and limitations of two independent screens that differed in the nature of the marker DNA used (PCR-amplified fragment containing the plasmid-free marker versus entire linearized plasmid with the marker) and in the strategies used to maintain and store transformants.

Figure 1

Primer design. Depicted is an actual case of the Chlamydomonas RDP3 target gene (ID, 183511) interrupted by the marker gene. The marker gene consisted of a PCR-amplified fragment containing the AphVIII gene under the control of the PSAD promoter, and with an incomplete 3'UTR. Since the DNA marker can be integrated into the Chlamydomonas genome in two different orientations, two forward (F1 and F2) and two reverse target gene primers (R1 and R2) were used to identify the insertion site. Typically, forward and reverse primers are separated by ~1.0 kb along the sequence of the target gene. Each target gene primer is used in combination with individual marker gene primers (RB1 and RB2) in independent PCR amplifications. Amplification would only be possible in those transformants in which the marker gene is inserted close to or within the target gene. In the example depicted, the marker gene was inserted within the 5'UTR of the target gene and amplification was observed with the RB2-R1 primer pair. Exons, introns and UTR regions are represented by black, white and grey squares respectively.

Figure 2

Work-flow diagram used for the reverse genetics approach. A. Generation and selection of transformants. Thousands of transformants are generated using the AphVIII marker gene and selected on paromomycin-containing plates. B. Isolation of individual transformants. Individual colonies are cultured in 96-well microtiter plates (200 μl per well). C. Transformant pooling. Aliquots (25-50 μl) from each well of an individual 96 well microtiter plate are pooled and cultured in fresh medium (pool). D. Isolation of genomic DNA from pools. DNA from each pool of transformants is isolated and diluted to 100 ng/μL. E. Generation of DNA superpools. Superpools are constructed by combining equal volumes of genomic DNA from 10 different pools. F. Screening the superpools. A set of independent PCR reactions using marker and target gene primers is performed with each DNA superpool as template (in the real case depicted, amplification occurred in the superpool sample loaded in lane 16) G. Confirmation of PCR products. Amplified PCR products are sequenced using the marker gene primer. H. Screening specific pools. The 10 different pools of DNA that comprise the superpool are individually screened using the appropriate primer pair. I. Screening of individual transformants. Positive transformants are identified within a specific microtiter plate by colony PCR. J. Isolation of specific transformants. Cells from the well containing the positive transformant are streaked onto solid medium to obtain single cell-derived colonies, which are then screened by colony PCR. This step is used to eliminate potential cross-contamination among transformants.

Figure 3

Determination of marker gene copy number by Southern blot hybridization. Southern-blot analysis of digested (PstI) genomic DNA from 9 randomly isolated paromomycin resistant mutants. A fragment of the AphVIII gene labeled with ³²P-dNTPs was used as probe. As shown, most transformants have a single copy of the integrated marker gene. Only the transformant represented in lane 4 may have two insertions (indicated by the two hybridizing bands). p.s., parental strain used to obtain the transformants.