A toolbox of Stable Integration Vectors (SIV) in the fission yeastSchizosaccharomyces pombe

Abstract

Schizosaccharomyces pombe is a widely used model organism that resembles higher eukaryotes in many aspects of cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research.

Activation of Cdc42 GTPase upon CRY2-Induced Cortical Recruitment Is Antagonized by GAPs in Fission Yeast

“…Gene tagging was performed at endogenous genomic locus at the 3′ end, yielding C-terminally tagged proteins, as described [ 35 ]. Pak1 gene tagging was performed by transforming a WT strain with AfeI linearized (pBSII(KS + ))-based single integration vector (pAV72-3′UTR pak1 -Pak1-sfGFP-kanMX-5′UTR pak1 ) targeting the endogenous locus [ 36 ]. The functional mCherry-tagged and sfGFP-tagged cdc42 alleles cdc42-mCherry sw and cdc42-sfGFP sw were used as described in [ 4 ].…”