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

Abstract

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.

Keywords: A/T-rich genome; Dictyostelium discoideum; Knockin; Linear DNA cloning.

Fig. 1

Knockin vector construction by a linear DNA cloning system. a 3-step construction of the 3′-tagging vector. Step 1: Preparation of pJAZZ vector harbouring A/T-rich 3′ recombination. Step 2: Assembly of the knockin vector by 4-piece ligation. Step 3: Release of knockin cassette by NotI digestion. b Design of GFP knockin vector for DDB_G0273397/carA-1 harbouring 1.0 kb each of 5′ and 3′ recombination arms. c, d Stable cloning of A/T-rich 3′ UTR/terminator of carA-1 by linear cloning system. 1 kb of 3′ UTR/terminator of carA-1 were blunt cloned into pBluescript (c) and pJAZZ vector (d). Release of the insert in randomly selected 6 DNA clones was checked by restriction enzyme digestion with XhoI and SpeI for pBluescript and with NotI for pJAZZ (these are the multiple cloning sites on each vector). Variable size of released fragments in C indicates deletions of circular plasmids. Appropriate size of inserts (arrow in d) were released from all the 6 clones of pJAZZ vector. The lane for negative control (Vector) was loaded with NotI-digested pJAZZ vector carrying no insert. Arrow heads represent the long and short arm of NotI-digested pJAZZ vector. e Four DNA fragments as depicted in B were subjected to directional ligation. f DNAs from randomly selected TSA E. coli clones were digested with NotI to excise the 4.5 kb of assembled knockin cassette (arrow). g Appropriate DNA assembly in 4 clones (same as in f) was detected by PCR for fragment ligation between 2 and 3 (upper column, 2 + 3) or fragment 3 and 4 (bottom column, 3 + 4). Primer position was depicted in (b). All the molecular marker was1 kb DNA ladder

Fig. 2

Generation of GFP knockin strain for carA-1. a Genomic organization of wild-type (WT) and GFP knockin locus for DDB_G0273397/carA-1. b WT and knockin locus before and after the removal of BsR cassette was detected by PCR. The primer set fw1/rev1, both located outside the homology arms, detects WT and knockin locus (pre_Cre) as 2.0 and 4.5 kb, respectively. BsR-removal was detected as the decrease in the size of target locus from 4.5 kb (pre-Cre) to 2.7 kb (post-Cre). Primer combination of rev2 for GFP and fw1 confirms specific recombination at the 5′ arm by yielding a 1.0 kb fragment. c Expression of GFP-tagged cARA-1 protein of the knockin strain detected by western blotting. Lysate of cells over-expressing cARA-1-GFP from extrachromosomal plasmid were loaded as the detection control (1/10 volume, cARA-1-GFP O.E)

Fig. 3

Triple-colour knockin for ACAA-GFP, REGA_mRFPmars and cARA-1-Turq2. a Genomic organization of wild-type (WT) and knockin locus (KI) for DDB_G0281545/acaA (left column), DDB_G0284331/regA (middle column) and DDB_G0273397/carA-1 (right column) tagged with green, red and cyan fluorescent proteins, respectively. Serial knockin in this order was performed, each followed by Cre-mediated BsR-recycling. Specific recombination was detected by PCR as in Fig. 2b. Primer fw1/rev1 detects a knockin event (WT to knockin_pre-Cre) and removal of BsR cassette (knockin_pre-Cre to post-Cre) as a 2.5 kb increase and 1.7 kb decrease of the amplified band, respectively. The fw1/rev2 set detects specific recombination at the 5′ recombination arm. b Protein expression of triple-colour knockin strain. Lysate from over-expressing cells (1/10 volume) with ACAA-GFP, REGA-mRFPmars and cARA-1-GFP were loaded as the positive control. c Live confocal images of the triple-colour knockin strain developed for 8 h. ACAA-GFP and cARA-1-Turq2 were localized at the cell surface. REGA-mRFPmars was detected in the cytoplasm excluded from the nucleus. Bar: 10 μm