Sequential and counter-selectable cassettes for fission yeast

Abstract

Background: Fission yeast is one of the most commonly used model organisms for studying genetics. For selection of desirable genotypes, antibiotic resistance cassettes are widely integrated into the genome near genes of interest. In yeasts, this is achieved by PCR amplification of the cassette flanked by short homology sequences, which can be incorporated by homology directed repair. However, the currently available cassettes all share the same tef promoter and terminator sequences. It can therefore be challenging to perform multiple genetic modifications by PCR-based targeting, as existing resistance cassettes in strains can be favored for recombination due to shared homology between the cassettes.

Results: Here we have generated new selection cassettes that do not recombine with those traditionally used. We achieved this by swapping the tef promoter and terminator sequences in the established antibiotic resistance MX6 cassette series for alternative promoters and/or terminators. The newly created selection cassettes did not recombine with the tef-containing MX6 cassettes already present in the genome, allowing for sequential gene targeting using the PCR-based method. In addition, we have generated a series of plasmids to facilitate the C-terminal tagging of genes with desired epitopes. We also utilized the anti-selection gene HSV-TK, which results in cell death in strains grown on the drug 5-Fluoro-2'-deoxyuridine (FdU, Floxuridin or FUDR). By fusing an antibiotic resistance gene to HSV-TK, we were able to select on the relevant antibiotic as well as counter-select on FdU media to confirm the desired genomic modification had been made. We noted that the efficiency of the counter selection by FdU was enhanced by treatment with hydroxyurea. However, a number of DNA replication checkpoint and homologous recombination mutants, including rad3∆, cds1∆, rad54∆ and rad55∆, exhibited sensitivity to FdU even though those strains did not carry the HSV-TK gene. To remove counter-selectable markers, we introduced the Cre-loxP irreversible recombination method. Finally, utilizing the negative selectable markers, we showed efficient induction of point mutations in an endogenous gene by a two-step transformation method.

Conclusions: The plasmid constructs and techniques described here are invaluable tools for sequential gene targeting and will simplify construction of fission yeast strains required for study.

Keywords: DNA replication; FUdR; Gene disruption and insertion; HA, Flag, PK tagging; Point mutation; Schizosaccharomyces pombe; Thymidine kinase; Zeocin.

Fig. 1

Selection markers for sequential gene targeting. a Schematic representation of sequential targeting cassettes pFA6a-neoCV, pFA6a-zeoCV, pFA6a-natCX, pFA6a-hygMV and pFA6a-hygML. The region used as a PCR template is shown. The 100 base Top (Tag) primer anneals to the left end (pink arrowhead on the left) and Bot primer anneals to the right end to amplify indicated cassettes (pink arrowheads on the right). The 100 base primers used in this study are listed in Table 1. Cyan arrows represent diagnostic primers used for screening of correct targeting (Table 2). Sequences of primers are shown in Table 2. Backbone vector region (pFA6a) encodes ampicillin resistant cassette and ColE1 bacteria replication origin, and the cassettes were inserted between PacI and PmeI sites. Black box and black arrow in the cassette represent indicated promoter and terminator, respectively. White box in neoCV and zeoCV indicates the em7 promoter for E. coli. The size of the cassette is shown on the left. Transcription direction is toward left for neoCV and zeoCV cassettes and toward right for other cassettes. b Wild-type and rdh54∆ cells in which the zeoCV cassette replaced rdh54 were cultured in YES rich media, normalised and serially diluted. Five microliter of diluted fractions were spotted on YES (input) or YES containing 100 μg/ml of Zeocin^TM and incubated at 32 °C for 3 days. Only cells containing the zeoCV cassette grew on YES with Zeocin. Deletion of the rdh54 gene required for meiosis does not impair cell growth and mitotic DNA damage repair. c Cells carrying kanMX6 or natMX6 cassettes were transformed with hygMV (Top) or hygMX6 (Bottom) and selected for on YES plates containing 100 μg/ml hygromycin (Hyg). Eighteen and 31 colonies that were randomly picked for hygMV and hygMX6 transformations respectively, were re-streaked on YES plates containing 100 μg/ml Hyg (Left panel). Cells were then replica-plated to YES plates containing 100 μg/ml Hyg plus either 100 μg/ml G418 (Middle panel) or 100 μg/ml ClonNat (Nat) (Right panel). In case of hygMV transformation, all transformed cells retained resistance to G418 and ClonNat, whereas in case of hygMX6, only seven out of 31 did

Fig. 2

TK-fusion counter-selectable cassettes. a Schematic representation of counter-selectable cassettes HyTKAX, TKanAX and TKnatAX. Region used as a PCR template is shown. The backbone vector is pFA6a and the cassettes were inserted between PacI PmeI sites. Top primers anneal to the left end (pink arrowhead on the left) and Bot primer anneals to the right end to amplify indicated cassettes (pink arrowheads on the right). Cyan arrows represent diagnostic primers used for screening of correct targeting, and sequence of primers is shown in Table 2. The TK fusion genes are transcribed by the S. pombe adh1 promoter and terminated at the tef terminator. The size of the cassette is shown on the left. Transcription direction is toward right. b, c and d FdU sensitivity of cells carrying the HSV-TK fusion cassettes. Indicated cells were cultured in YES rich media, normalised and sequentially diluted five times. Five microliter of diluted fractions were spotted on YES containing the indicated drugs and incubated at 32 °C for 3 days. b Cells carrying the TKnatAX cassette are hyper sensitive to 20 μl/ml FdU. c Cells carrying the TKanAX cassette are sensitive to 100 μl/ml FdU. d Cells carrying the HyTKAX cassette are sensitive to 20–100 μl/ml FdU

Fig. 3

Concomitant treatment with HU enhances sensitivity to FdU. Indicated cells were cultured in YES, normalised and serially diluted five times with YES. Five microliter of diluted fractions were spotted on rich media containing the indicated drugs and incubated at 32 °C for 3 days. Cells carrying TKanAX grew poorly in YES containing 5 mM HU and 100 μg/ml FdU

Fig. 4

rad3∆, cds1∆ and rad54∆ are sensitive to FdU. Indicated cells were cultured in YES, normalised and 5 times sequentially diluted with YES. Five microliter of diluted fractions were spotted on rich media containing the indicated drugs and incubated at 32 °C for 3 days. a rad3∆ cells are hyper sensitive to 20 μg/ml FdU. b cds1∆ and rad54∆ cells are sensitive to 100 μg/ml FdU. chk1∆ and rad55∆ cells are mildly but significantly sensitive to FdU, compared to wild type cells

Fig. 5

Cre expression vector and loxP cassette. a Schematic representation of loxP-flanked (floxed) TK-fusion cassettes FHyTKAX, FTKanAX and FTKnatAX. Region used as a PCR template is shown. Forward direction of lox71 and lox66 sequences are inserted into the TK cassettes shown in Fig. 2a. b Schematic representation of pNXRVa-HACre. Fusion protein of three tandem HA epitope tag, NLS (nuclear localization signal) and Cre recombinase is expressed by the constitutively active CMV promoter. kanMX6 is inserted between BglII and PmeI sites. The genomic DNA fragment containing the early replication origin ARS1 is inserted downstream of the kanMX6 cassette. A number of unique restriction enzyme sites are indicated

Fig. 6

The COOH-terminus tagging plasmid. a Schematic representation of pNX3a-HA3 and sequence of three tandem HA and multi cloning sites. The HA encoding gene is inserted between PacI AscI sites. The 100 base Tag primer anneals to the left end (pink arrowhead on the left in plasmid image and long arrow in HA3 sequence) and Bot primer anneals to the right end to amplify indicated cassettes (pink arrowheads on the right in plasmid image). A number of unique restriction enzyme sites are indicated. b Sequence of three tandem PK (top) and FLAG (bottom) fragments. pNX3a-PK3 and pNX3a-FL3 plasmids were generated by replacing HA3 sequence between NheI and XbaI sites in pNX3a-HA3 (a) with indicated PK and FLAG sequences, respectively. c Detection efficiency of PK-tagged Tpz1. Western blot shows detection of PK epitope fused Tpz1. No obvious non-specific bands were detected. Proteins were extracted from cells and subjected to SDS-PAGE. Anti-V5 antibody (AbD Serotec) was used to detect PK fused Tpz1 protein. Anti-Cdc2 antibody (anti-PSTAIRE) (Santa Cruz) was used as a control for loading. d Telomere length homeostasis is slightly impaired with the nine tandem PK tagging of Tpz1. Genomic DNA was harvested from cells cultured over 2 weeks after generation of strains, and digested with EcoRI and separated in 1 % agarose gel. Telomere containing fragments were detected with the synthetic telomeric DNA probe

Fig. 7

Two-step mutagenesis. Schematic diagram of the two-step gene replacements and induction of point mutation of the tpz1 gene. List of diagnostic primers is shown in Table 2. a PCR based tpz1 gene deletion by the TKnatCX targeting fragment generated using the tpz1 Top and Bot 100 base primer set (Table 1). 80–100 base of homologous sequences in the 100 base primer set targets 5’ and 3’ UTR regions of tpz1 ⁺ of one of endogenous tpz1 ⁺ alleles. Replacement of tpz1 ⁺ by TKnatCX was confirmed by amplification of DNA fragment using diagnostic PCR primers, nat F900 and tpz1 R400D. b Replacement of the TKnat cassette by the tpz1 mutant gene. The DNA fragment containing the promoter region and the gene of tpz1 ⁺ was amplified using primers tpz1 F800U and R1745 and cloned between EcoO109I and NheI sites of pNX3a-HA3. The tpz1 gene was mutated to generate K75A mutation using the sight-directed mutagenesis method. PvuII and PmeI digested tpz1(K75A)-3xHA:kanMX6 fragment from pTpz1a4-K75A-HA3 can only recombine with TKnatCX deleted tpz1 allele, as right arm homology Ttef sequence is only present in the TKnatCX cassette. c Integration of tpz1-K75A mutation and 3xHA tagging. Correct replacement was confirmed by amplification of DNA fragments using diagnostic PCR primer sets, kan F800 and tpz1 R400D, and tpz1 F842U and Ptef R81 (or kan R276)