Related Experiment Video
Updated: Sep 23, 2026
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Published on: July 12, 2013
In vitro generation of cloned populations of Toxoplasma gondii
C Sreekumar1, J R Rao, A K Mishra
1Division of Parasitology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243 122 Uttar Pradesh, India. kumar@anri.barc.usda.gov
Abstract:
Cloned populations were generated from Indian isolates of Toxoplasma gondii by transferring single tissue cysts from the brains of chronically infected mice to confluent murine macrophage (J774A.1) monolayers. The clones were then maintained continuously as tachyzoites in culture. Physical rupture of the tissue cysts and release of bradyzoites prior to seeding was found to be necessary for establishment of the parasite in culture. Although intact tissue cysts seeded over monolayers released bradyzoites spontaneously, they did not succeed in setting up an infection in the monolayers. Random amplified polymorphic DNA (RAPD)-PCR, which revealed distinct patterns for a clone and its progenitor, further confirmed the efficiency of the technique. The cloning technique was found to be simple and rapid compared to those involving limiting dilutions.
Insights
Generating cloned Toxoplasma gondii populations from Indian isolates requires physically rupturing tissue cysts to release bradyzoites. This method is faster than traditional limiting dilution techniques for parasite culture.
Area of Science:
- Parasitology
- Molecular Biology
- Infectious Diseases
Background:
- Toxoplasma gondii is a significant protozoan parasite.
- Generating cloned parasite populations is crucial for research.
- Indian isolates of T. gondii require specific isolation techniques.
Purpose of the Study:
- To develop and validate a rapid and efficient method for cloning Indian isolates of Toxoplasma gondii.
- To establish a continuous tachyzoite culture from cloned bradyzoites.
- To confirm the genetic integrity of cloned parasite populations.
Main Methods:
- Cloning T. gondii from infected mouse brain tissue cysts.
- Culturing tachyzoites on murine macrophage (J774A.1) monolayers.
- Utilizing physical rupture of tissue cysts to release bradyzoites.
- Employing Random Amplified Polymorphic DNA (RAPD)-PCR for genetic analysis.
Main Results:
- Successful generation of cloned T. gondii populations from Indian isolates.
- Establishment of continuous tachyzoite cultures requires pre-ruptured tissue cysts.
- Intact tissue cysts did not initiate infection in macrophage monolayers.
- RAPD-PCR confirmed distinct genetic profiles between clones and progenitors, validating the cloning efficiency.
Conclusions:
- Physical rupture of T. gondii tissue cysts is essential for initiating parasite culture.
- The described cloning technique is simple, rapid, and effective.
- This method facilitates the study of genetically uniform T. gondii isolates.
