Fig. 3

Targeted disruption of the mlf gene resulted in decreased expression of the cwp1 and cwp2 genes during vegetative growth using strategy 4. A Schematic presentation of the pgCas9 and pNMLFtd plasmids. In construct pgCas9, the cas9 gene is flanked by gdh promoter (striated box) and 3′ untranslated region of the ran gene (dotted box). The nuclear localization signal (filled gray box) and an HA tag (filled black box) are fused to the C terminus. In construct pNMLFtd, a single gRNA is under the control of the Giardia U6 promoter. pNMLFtd also has the HR template cassette composed of the neo selectable marker and the 5′ and 3′ flanking region of the mlf gene as homologous arms. The Cas9/gRNA cutting site in the genomic mlf gene is indicated by a red arrow. Replacement of the genomic mlf gene with the neo gene will occur by HR, after introducing a double-stranded DNA break in the mlf gene. The pgCas9 and pNMLFtd constructs were transfected into trophozoites. The MLFtdNeo stable transfectants were established under G418 selection. G418 was removed from the MLFtdNeo cell line to obtain the MLFtdNeo –G418 (MLF KD) cell line. The control cell line is wild-type nontransfected WB trophozoites. To complement the targeted disruption of the mlf gene, a pPMLF expression vector was transfected to the MLF KD cell line. pPMLF is also described in Fig. 1A. The MLF KD + MLF (Complement) cell line was established under puromycin selection to maintain the MLF expression cassette. The control cell line is MLF KD cell line transfected with 5’∆5N-Pac plasmid and selected with puromycin. B PCR confirmed partial replacement of the mlf gene with the neo gene in the MLF KD cell line. Genomic DNA was isolated from MLF KD and control cell lines cultured in growth medium (vegetative growth, Veg). PCR was performed using primers specific for mlf (PCR1 in panel A), neo (PCR2 in panel A), cwp1, cwp2, and ran genes, respectively. Products from the cwp1, cwp2, and ran genes are internal controls. C Real-time PCR confirmed partial disruption of the mlf gene in the MLF KD cell line. Real-time PCR was performed using primers specific for mlf, cwp1, cwp2, and ran genes, respectively. The mlf, cwp1, and cwp2 DNA levels were normalized to the ran DNA level. The ratio of DNA levels in MLF KD cell line to levels in control cell line is shown and expressed as the means ± 95% confidence intervals of at least three separate experiments. **, p < 0.01. ns, p > 0.05, not significant. D Targeted disruption of the mlf gene in the MLF KD cell line resulted in decreased cyst generation during vegetative growth. Cyst number was counted from the control and MLF KD cell lines cultured in growth medium. Fold changes in cyst generation are shown as the ratio of the sum of total cysts in the MLF KD cell line relative to the control cell line. Values are shown as mean ± 95% confidence intervals. **, p < 0.01. E The CWP1 protein level decreased by targeted disruption of the mlf gene in the MLF KD cell line during vegetative growth. The control and MLF KD cell lines cultured in growth medium were subjected to SDS-PAGE and Western blot analysis using anti-MLF, anti-CWP1, and anti-RAN antibodies, respectively. SDS-PAGE with Coomassie Blue staining is included as a control for equal protein loading. The band intensity from triplicate Western blots was quantified using Image J as described in Fig. 1E. *, p < 0.05. F Targeted disruption of the mlf gene in the MLF KD cell line resulted in decreased expression of cwp1 and cwp2 during vegetative growth. The control and MLF KD cell lines cultured in growth medium were subjected to quantitative real-time RT-PCR analysis using primers specific for mlf, cwp1, cwp2, ran, and 18S ribosomal RNA genes, respectively, as described in Fig. 2C. ***, p < 0.001. ns, p > 0.05, not significant