Generation of conditional knock out mutants for Tgndh2-I

In order to study the link between carbohydrate metabolism and stage conversion as well as the role of respiratory chain components on the growth of the parasites, mutant parasites which are deficient in the following genes: (i) ndh2-I, (ii) sdh-fp, (ii) cytc1, and (iv) ATP-β, encoding for key subunits of the respiratory chain were seeked to be generated by targeted gene disruption.

Two strategies were applied for generation of these mutants. The first aims to generate a knock-out mutants by using a targeting construct harbouring 2 inserts flanking a CAT expression cassette as selectable marker (double homologous recombination) (Fig. 3.22) or by using targeting construct with single insert and CAT selectable marker (single homologous recombination) (Fig. 3.23). Parasites expressing the additional copies of the targeted genes, under the control of Tet-dependent transactivator promoter, were used to generate conditional knockout mutants (Fig.

3.24).

Figure 3.22: Targeting scheme for generation of a conditional knock-out mutants by double homologous recombination. Two fragments of the targeted gene of ~2.5 kb, about 100-200 bp away from each other, were cloned in pKS-CAT vector flanking the CAT expression cassette. A double cross-over event at the endogenous locus results in the integration of the targeting construct (F1-CAT-F2) into the targeted gene and generates a non-functional gene. Red arrows indicate the positions of primer sequences used for subsequent PCR analysis.

RESULTS 84.

Targeting constructs and T. gondii clones expressing the exogenous copies of the targeted genes (designated; ndh2-i/ndh2-i-cmyc, ndh2-ii/ndh2-ii, sdh-fp/sdh-fp-cmyc, cytc1/cytc1-cmyc, and ATP-β/ ATP-β-cmyc) (Fig 3.8) were generated as described in chapter 2 (Materials and Methods). Clones which displayed a tight Atc-dependant regulation of the 5 target genes were chosen for deletion of the endogenous copies of these genes, the parasites were stably transfected with a targeting vectors containing single and double inserts to allow for the disruption the endogenous copies of the genes by homologous recombination. Individual clones were screened by PCR for loss of the wild type alleles and for integration of the targeting constructs by homologous recombination in the endogenous locus of the gene. All tissue cultures were performed without Atc, in order to induce the expression of the exogenous copies of the target genes. Unfortunately, the PCR screening for the stably transfected parasites with both strategies did not show disruption of the endogenous genes coding for TgNDH-II, TgSDH-Fp, TgCYTC1 and TgATP-β, most likely due to the high frequency of

non-3+

Figure 3.23: Generation of a conditional knock-out mutants by single homologous recombination. Parasites expressing exogenous copies of the targeted gene were transfected with a circular targeting vector, containing a 2.5 kb genomic fragment and a CAT expression cassette (TUB/CAT). The 2.5 kb genomic fragment encodes for a truncated, non-functional gene which lacks essential N- and C-terminal sequences of the coding region. A single cross-over event at the endogenous locus results in the integration of the targeting plasmid into the endogenous gene and generates two non-functional allels. Arrows indicate the positions of primer sequences used for subsequent PCR analysis.

RESULTS 85.

specific integration of the targeting vectors by non-homologous recombination.

Regarding the gene coding for TgNDH2-I, which was targeted by a construct with single DNA insert, one clone out of about 120 was identified to display the expected pattern of PCR products for the knock out (Fig. 3.25a). Nested PCR was used to confirm the specificity of the PCR products (Fig. 3.25b). RT-PCR analysis confirmed the absence of ndh2-I mRNA from the endogenous ndh2-I gene, demonstrating the functional disruption of the ndh2-I locus (Fig. 3.25c). The knock out parasites (∆ndh2-i/ndh2-i-myc) were further used for phenotypic analysis.

Figure 3.24: Tetracycline inducible transactivator system (A) Schematic representation of the tetracycline inducible transactivator system (tet off) in T. gondii expressing TATi. (B) Atc-regulated expression of AND1-myc. AND1-myc expression was detected by immunofluorescence microscopy in andh1/Andh1-myc parasites using anti-myc mAb 9E10.

Andh1/Andh1-myc parasites were cultured one round before either in the absence or presence of Atc. AND1-myc expression was only detectable in the absence (B1), but not in the presence of Atc (B2).

A.

T. gondiiline expressing TATi (Meissner et al. 2002) Tet-repressor Transactivating domain

RESULTS 86.

M A B C M A B C A B C uncloned

A.

C.

bp 500 250

B.

ndh2-i/Ndh2i-cmyc ∆ndh2-i/Ndh2i-cmyc

∆ndh2-i /ndh2i-cmyc

ndh2-i/

ndh2i-cmyc

1.5 1.0 0.75 -kb 3.0 2.5 2.0 -kb

ndh2i tub ndh2i tub ndh2i tub ndh2i tub

| | | | | | | |

Figure 3.25: Conditional knock out of TgNDH2-I. (A) Confirmation of Δ andh1/Andh1-myc identity by PCR. Primer pairs, which are specific for the intact wild type allele (A) and for the two non-functional and1 allels (B and C) were used for PCR analysis on genomic DNA of (i) an uncloned, stabely transfected population, (ii) a single Δandh1/Andh1-myc clone and (iii) parental strain (andh1/Andh1-myc) parasites. Primers used for amplification of fragment “A” were: AND1_ 3+/AND1_4-; for fragment “B”:

AND1_ 3+/T7-; for fragment “C”: SAG1+/AND1_ 4-. Δandh1/Andh1-myc parasites displayed the expected absence of the wild-type specific band and the presence of the two non-functional allels. (B) The specificity of PCR products “B” and “C” was confirmed by nested PCR using various internal primer combinations (see Materials and Methods). (C) RT-PCR analysis on Δandh1/Andh1-myc parasites for andh1 mRNA expression. Parental strain (ndh2-I/Ndh2-I-myc) and Δndh2-I/Ndh2-I-myc parasites were cultured either with or without Atc for several rounds. cDNA of these parasites was amplified with a ndh2-I specific primer pair which amplifies both, the endogenous and the ectopic copy. Tubulin (tub) primers served as a control. The absence of a ndh2-I PCR product in Δ andh1/Andh1-myc parasites cultured with Atc demonstrates both, the lack of ndh2-I mRNA derived from the endogenous gene and the successful repression of the ectopic copy.

RESULTS 87.

3.3.2 Phenotypic analysis of knock out parasites (

Δ