A rapid and efficient method to generate multiple gene disruptions in Dictyostelium discoideum using a single selectable marker and the Cre-loxP system

Abstract

Dictyostelium discoideum has proven an exceptionally powerful system for studying numerous aspects of cellular and developmental functions. The relatively small ( approximately 34 Mb) chromosomal genome of Dictyostelium and high efficiency of targeted gene disruption have enabled researchers to characterize many specific gene functions. However, the number of selectable markers in Dictyostelium is restricted, as is the ability to perform effective genetic crosses between strains. Thus, it has been difficult to create multiple mutations within an individual cell to study epistatic relationships among genes or potential redundancies between various pathways. We now describe a robust system for the production of multiple gene mutations in Dictyostelium by recycling a single selectable marker, Blasticidin S resistance, using the Cre-loxP system. We confirm the effectiveness of the system by generating a single cell carrying four separate gene disruptions. Furthermore, the cells remain sensitive to transformation for additional targeted or random mutagenesis requiring Blasticidin selection and for functional expression studies of mutated or tagged proteins using other selectable markers.

Figure 1

A strategy for Cre-loxP recycling of the Bsr selectable marker. (A) The loxP recombination site includes an inverted repeat separated by a spacer sequence. (B) The gene targeting vector pLPBLP was constructed with loxP sites in the same orientation flanking both sides of the Bsr expression cassette act15/Bsr. An oligonucleotide cassette was also added that had translational stop codons in all six reading frames. The restriction enzyme sites outside of the floxed-Bsr cassette, such as SmaI (see Materials and Methods), permits the cloning of 5′ and 3′ gene sequences for targeted disruption. (C) The floxed-Bsr cassette of pLPBLP was inserted into the gene DDB0183838 sequence as indicated (see also Figure 2A). A BamHI site was engineered into the targeted gene sequence. This was filled and blunt-end ligated to the SmaI sites flanking the floxed-Bsr cassette to generate the gene-targeting vector. WT cells were transformed for gene disruption by homologous recombination and selected for resistance to Blasticidin S. (D) Transient expression of Cre promotes recombination and deletion of sequences between the two loxP sites in the disrupted gene DDB0183838. A 73 nt sequence remains that includes the translational stop cassette and a single loxP site. The sequence presented was determined directly from a PCR-amplified fragment of the region (see Figure 2B) and matches the predicted recombination event. The disrupted target gene sequences are in green, the loxP site is underlined and the stop codons are indicated by arrows. The site of the BamHI/SmaI fusion is also indicated.

Figure 2

Cre recombination deletes the Bsr selectable marker. (A) Genomic sequences flanking the insertion site of gene DDB0183838 depicted in Figure 1C were amplified by PCR and analyzed by agarose gel electrophoresis. WT is the endogenous wild-type gene sequence of ∼450 bp. Knockout mutants (KO) are clonal isolates of cells transformed and selected for resistance to Blasticidin S; insertion of the floxed-Bsr cassette yields a fragment of ∼2 kb. (B) A KO isolate from Figure 2A was transformed for transient expression of Cre, and several clonal isolates were randomly screened for functional recombination by PCR as in Figure 1A. Wild-type is the endogenous WT gene sequence of ∼450 bp. KO is the gene sequence carrying the insertion of the floxed-Bsr cassette (∼2 kb). Recombination between loxP sites generates a fragment that is ∼520 bp. The fragment was sequenced for confirmation (see Figure 1D). (C) Genomic DNA from WT, KO and Cre cells were probed by southern blot hybridization for the presence of Bsr and Neo gene sequences. DNA from a Neo-expressing transgenic cell line was included as a control.

Figure 3

Cre-recombined cells are susceptible to additional Bsr recycling. (A) A Cre-recombinant isolate from Figure 2B was transformed for a secondary gene disruption within DDB0184318 and selected for resistance to Blasticidin S. Genomic sequences flanking the insertion site of gene DDB0184318 were examined by PCR amplification. WT is the endogenous wild-type gene sequence; KO mutants are selected clonal isolates with the insertion of the floxed-Bsr cassette. (B) A KO isolate from Figure 3A was transformed for transient expression of Cre, and several clonal isolates were randomly screened for functional recombination by PCR as in Figure 2A. KO is the gene sequence carrying the insertion of the floxed-Bsr cassette. Recombination between loxP sites generates a fragment that is only ∼70 bp larger than the WT sequence.

Figure 4

Strategy for the generation of a quadruple drf-null cell line. (A) Generation of a drf1-null cell line. The 5′ and 3′ specific sequences of the drf1 gene were cloned into pLPBLP. The linear targeting vector was then used to disrupt the drf1 gene by homologous recombination. Targeted integration also caused a small deletion in the gene. A similar approach was used to construct targeting vectors for drf genes 2, 3 and 4. (B) WT cells and three independent Bsr mutants were examined by PCR amplification employing two different sets of drf1 primers. Left panel: the primer combination of U1 and D1 (see A) identifies WT and homologously recombined drf1 sequences. Upon homologous recombination, the drf1 PCR product is ∼1.3 kb larger than that of WT for all three cell lines shown. Right panel: the primer combination of Bsr and D1 (see A) specifically identifies only the homologous recombination event. (C) Strategy for deletion of Bsr by transient expression of NLS-cre. Deletion of the floxed-Bsr cassette leaves a sequence of ∼70 nt. (D) PCR analysis of drf1-null derived cells following transient expression of NLS-cre (^*). All three clonal cell lines that were sensitive to Blasticidin and G418 for growth and were also deleted of the floxed-Bsr cassette. (E) Elimination of three additional drf genes. Top panel: three additional alternating rounds of gene inactivation followed by the subsequent elimination of the floxed/Bsr-cassette through transient expression of NLS-cre were performed to obtain a single mutant cell line lacking four drf genes. Each PCR screen used a common Bsr upstream primer and a downstream primer that was specific to drf genes 2, 3 or 4 (see A). Bottom panel: DNA from WT cells and the quadruple mutant 1^*/2^*/3^*/4^* was amplified by PCR using U and D primers specific to each drf gene to illustrate the stability of the four single loxP sites in the genome of the quadruple mutant. Each of the original homologous recombination events was designed to delete sequences from the endogenous gene (see A). Following the Cre-mediated recombination, an additional ∼70 bp remains that includes the translational stop cassette and a single loxP site (see Figures 1 and 4C), but still, the PCR products amplified from each gene of the 1^*/2^*/3^*/4^* mutant are always smaller than that of the WT gene fragment.