Fig. 1
From: Sequential and counter-selectable cassettes for fission yeast
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 ZeocinTM 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