The Cre/loxP system in Giardia lamblia: genetic manipulations in a binucleate tetraploid protozoan

Wampfler, Petra B.; Faso, Carmen; Hehl, Adrian B.

doi:10.1016/j.ijpara.2014.03.008

Cited by 19 publications

(20 citation statements)

References 43 publications

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“…This was combined with removal of a puromycin-resistance expression cassette ( PAC ) flanked by lox sites via Cre-mediated excision analogous to a previously demonstrated proof-of-concept1114 (see also in the section ‘Materials and Methods: Construction of CWP1KO1-4 plasmids and engineering of line Δcwp1 ' and Fig. 1a).…”

Section: Resultsmentioning

confidence: 99%

“…Genetic manipulation of Giardia has been established for over two decades with first attempts by electroporation and selection of circular plasmid episomes28, followed by strategies using Giardia virus RNA as a vector2930, and finally integration of linear vector DNA for modification of chromosomal DNA143132. Recently, genome engineering using the Cre/ loxP system has allowed elimination of a single allele followed by the removal of the selection marker from the chromosomal DNA11. In the attempt to perform functional characterizations of target genes, tools for depletion of target mRNA stability and/or inhibition of translation were developed and adapted to Giardia 3334.…”

Section: Discussionmentioning

confidence: 99%

“…In a previous report, proof-of-concept for the application of the Cre (Causes Recombination)/ loxP (locus of crossing (x) over, P1) system to obtain selection marker-free, transgenic G. lamblia lines carrying insertions at defined genomic loci was presented11. However, we found that chromosomal insertion of a linearized construct by double cross-over occurs only at a single locus in a transfection experiment.…”

mentioning

confidence: 84%

“…3a). These feature a puromycin-resistance expression cassette ( PAC ) driven by a constitutively-active promoter flanked by two loxP sites in the same orientation11. Upstream and downstream of this ‘floxed' PAC cassette we cloned ca.…”

Section: Methodsmentioning

confidence: 99%

“…PCR analysis of gDNA was performed as previously reported11. 3′RACE (rapid amplification of cDNA 3′ ends) was done as reported previously4.…”

Section: Methodsmentioning

confidence: 99%

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Cyst-Wall-Protein-1 is fundamental for Golgi-like organelle neogenesis and cyst-wall biosynthesis in Giardia lamblia

et al. 2016

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The genome of the protozoan parasite Giardia lamblia is organized in two diploid nuclei, which has so far precluded complete analysis of gene function. Here we use a previously developed Cre/loxP-based knock-out and selection marker salvage strategy in the human-derived isolate WB-C6 to eliminate all four copies of the Cyst-Wall-Protein-1 locus (CWP1). Because these loci are silenced in proliferating trophozoites and highly expressed only in encysting cells, CWP1 ablation allows functional characterization of a conditional phenotype in parasites induced to encyst. We show that encysting Δcwp1 cells are unable to establish the stage-regulated trafficking machinery with Golgi-like encystation-specific vesicles required for cyst-wall formation but show morphological hallmarks of cyst development and karyokinesis. This ‘pseudocyst' phenotype is rescued by transfection of Δcwp1 cells with an episomally maintained CWP1 expression vector. Genome editing in genera Giardia and Trypanosoma are the only reported examples addressing questions on pathogen transmission within the Excavata supergroup.

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Section: Resultsmentioning

confidence: 99%