Reliable handling of highly A/T-rich genomic DNA for efficient generation of knockin strains of Dictyostelium discoideum.

BACKGROUND: Social amoeba, Dictyostelium discoideum, is a well-established model organism for studying cellular physiology and developmental pattern formation. Its haploid genome facilitates functional analysis of genes by a single round of mutagenesis including targeted disruption. Although the efficient generation of knockout strains based on an intrinsically high homologous recombination rate has been demonstrated, successful reports for knockin strains have been limited. As social amoeba has an exceptionally high adenine and thymine (A/T)-content, conventional plasmid-based vector construction has been constrained due to deleterious deletion in E. coli. RESULTS: We describe here a simple and efficient strategy to construct GFP-knockin cassettes by using a linear DNA cloning vector derived from N15 bacteriophage. This allows reliable handling of DNA fragments whose A/T-content may be as high as 85 %, and which cannot be cloned into a circular plasmid. By optimizing the length of recombination arms, we successfully generate GFP-knockin strains for five genes involved in cAMP signalling, including a triple-colour knockin strain. CONCLUSIONS: This robust strategy would be useful in handling DNA fragments with biased A/T-contents such as the genome of lower organisms and the promoter/terminator regions of higher organisms.

High-affinity tuning of single fluorescent protein-type indicators by flexible linker length optimization in topology mutant.

Genetically encoded Ca2+ indicators (GECIs) are versatile for live imaging of cellular activities. Besides the brightness and dynamic range of signal change of GECIs, Ca2+ affinity is another critical parameter for successful Ca2+ imaging, as the concentration range of Ca2+ dynamics differs from low nanomolar to sub-millimolar depending on the celltype and organism. However, ultrahigh-affinity GECIs, particularly the single fluorescent protein (1FP)-type, are lacking. Here, we report a simple strategy that increases Ca2+ affinity through the linker length optimization in topology mutants of existing 1FP-type GECIs. The resulting ultrahigh-affinity GECIs, CaMPARI-nano, BGECO-nano, and RCaMP-nano (Kd = 17-25 nM), enable unique biological applications, including the detection of low nanomolar Ca2+ dynamics, highlighting active signaling cells, and multi-functional imaging with other second messengers. The linker length optimization in topology mutants could be applied to other 1FP-type indicators of glutamate and potassium, rendering it a widely applicable technique for modulating indicator affinity.