Home › Genetics / Genomics › Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Genetics / Genomics JoVE (Open Access) Citable · DOI

Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii

DOI: 10.3791/50598-v
What you'll learn
  • ✓ Design and prepare targeting DNA plasmids for Toxoplasma gondii gene manipulation
  • ✓ Execute transfection and selection protocols in Δku80 strains
  • ✓ Generate targeted gene deletions and C-terminal tags using homologous recombination
  • ✓ Remove selectable markers for functional genomic analysis
Protocol

Here we report a method for using type I and type II Δku80 strains of Toxoplasma gondii to efficiently generate targeted gene deletions and gene replacements for functional genomic analysis.

Difficulty
advanced
Total time
~3–4 weeks (includes parasite culture, transfection, selection, and clonal expansion)
Model organism
Toxoplasma gondii (type I and type II Δku80 strains)
Biosafety
BSL-2

Steps

1
Prepare targeting DNA plasmid pGOI

Design and construct the targeting plasmid containing homology arms flanking the gene of interest and selectable marker. Verify plasmid sequence and integrity before transfection.

▶ 01:58
2
Transfect T. gondii parasites with targeting plasmid

Introduce the pGOI plasmid into type I or type II Δku80 strain parasites using electroporation or other transfection methods. Maintain parasite viability during the procedure.

▶ 02:48
3
Select transfected T. gondii parasites

Culture transfected parasites under selective pressure using the marker (e.g., HXGPRT) to enrich for clones with integrated targeting DNA. Isolate and expand individual clonal populations.

▶ 03:43
4
Delete HXGPRT marker and perform C-terminal tagging

Excise the selectable marker and introduce epitope tags (e.g., HA, FLAG) at the C-terminus of the target gene via secondary transfection. Verify modifications by PCR and Western blot.

▶ 07:07
5
Validate gene deletion and marker removal

Confirm successful deletion of target genes (e.g., rop18) and removal of HXGPRT marker using molecular and phenotypic assays. Document results for functional analysis.

▶ 07:48
💬 Comments coming soon