Rapid Tagging and Integration of Genes in Giardia intestinalis

Abstract

We developed a series of plasmids that allow C-terminal tagging of any gene in its endogenous locus in Giardia intestinalis, with different epitope tags (triple hemagglutinin [3HA] and triple Myc [3Myc]) and selection markers (puromycin, neomycin, and a newly developed marker, blasticidin). Using these vectors, cyclin B and aurora kinase were tagged, expressed, and localized.The protozoan Giardia intestinalis is a human parasite responsible for considerable morbidity both in humans and in cattle (1,9,15). Due to its high divergence from other eukaryotes, many antibodies useful for cell biology will not recognize Giardia proteins. The process of developing gene-specific antibodies is long and laborious, typically requiring several months. A faster approach is to express a version of the gene tagged with a well-characterized epitope by integrating a construct into the chromosome. Integration of constructs into the Giardia intestinalis genome has been achieved previously (6, 7, 11) but not for the purpose of tagging a gene. Our goal was to facilitate integration of a tagged Giardia gene into the genome and to keep expression as close to endogenous levels as possible.A construct was designed for knocking in a tagged version of cyclin B (GiardiaDB identification [ID] number GL50803_ 3977) into the Giardia genome. This strategy is derived from the gene tagging originally developed with Schizosaccharomyces pombe (2, 4) and since then adapted to other organisms, such as Trypanosoma brucei (10). The C-terminal part of the gene of interest is cloned in frame to an epitope tag in a vector also containing a selectable marker cassette. This region should contain a restriction enzyme site, preferably naturally occurring, that will be unique in the resulting vector; additionally, this site should be located at least 90 bp from either the 5Ј or 3Ј cloning site. The resulting plasmid is then linearized using this restriction enzyme and introduced into cells, and transfected cells are selected using the selectable marker. Single-site homologous recombination and integration of this construct should yield two copies of the gene (Fig. 1A shows more details): one under the control of the original promoter but now with a C-terminal epitope, the other missing any promoters and its entire N terminus and thus presumably not expressed.To tag Giardia cyclin B, an XhoI site was engineered by PCR to eliminate the cyclin B stop codon (see the supplemental material for oligonucleotide details). A StyI/XhoI cyclin B fragment was cloned in frame to a C-terminal triple-hemagglutinin (HA) epitope tag (3HA) (Fig. 1A) and appended to the NotI/KpnI fragment from the plasmid p␥G-GFP (11) containing the puromycin resistance cassette to yield the plasmid pc-cycB-3HA-PAC. The plasmid was then linearized using an original NruI site, ethanol precipitated, and resuspended in distilled water, and 10 g of linear DNA was introduced into Giardia strain WB-C6 by electroporation as previously described (11). Transfected cells were selected using puromycin (10 g/...

The Giardia cell cycle progresses independently of the Anaphase Promoting Complex

“…To investigate the function of Giardia cyclin B we used a Giardia cell line carrying a 3HA tagged allele of cyclin B (Gourguechon and Cande, 2011) and morpholino-mediated gene specific knockdown (Carpenter and Cande, 2009). Morpholino directed against the cyclin B mRNA efficiently reduced the levels of Giardia cyclin B to ,40% after 24 hours (Fig.…”

“…The gene for cyclin B (accession GL50803_3977) was amplified by PCR to introduce a 59 NdeI site and a 39 SmaI site. The NdeI/SmaI cyclin B fragment and a SmaI/BamHI fragment carrying the 3 myc tag [from vector pKS-3Myc-BSR (Gourguechon and Cande, 2011)] were cloned into NdeI and BamHI sites of pRep42. The resulting pRep42-cyclinB vector was transformed into fission yeast by electroporation.…”

“…The Giardia strain carrying an integrated 3HA tagged cyclin B allele was used (Gourguechon and Cande, 2011). For morpholino-mediated knockdown, the sequence of cyclin B was submitted to the Gene Tools LLC website (www.genetools.com) to design and obtain oligos.…”

“…Cell lines were constructed, using previously described methods (Gourguechon and Cande, 2011), to express a 3HA-tagged allele of AUK (accession GL50803_5358) or PLK (accession GL50803_104150). These cells were then incubated for 12 hours in the presence, or absence, of the proteasomal inhibitor MG262 (5 mM, Boston Biochemicals), then lysed in PBS pH 7.4 supplemented with Halt protease inhibitors (Thermo Fisher scientific).…”

“…The 3HA tag in pcCycB3HAPAC (Gourguechon and Cande, 2011) was replaced with the SNAP tag from pSNAPf (New England Biolabs) and the resulting vector, verified, linearized with NruI and transfected into Giardia cells as described. Cells expressing SNAP-tagged cyclin B (,1610 8 cells) were iced and harvested, then incubated in 1 ml of complete media containing 5 mM of SNAP-fluorescein (New England Biolabs) for 3 hours at 37˚C.…”