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Updated: Jun 3, 2026
09:52
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Published on: July 12, 2013
[Construction and functional analysis of a common gene targeting vector with double-selection markers]
Junhua Li1, Cuiqin Han, Jie Deng
1Shaanxi Key Laboratory of Molecular Biology for Agriculture, Shaanxi Center for Stem Cell Engineering and Technology, College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China.
Summary
Researchers developed an efficient gene targeting vector, pGT-V1, for mammalian genome modification. This vector enhances gene knockout efficiency and allows for marker removal, aiding transgenic animal research.
Area of Science:
- Molecular Biology
- Genetics
- Genomics
Background:
- Homologous recombination is crucial for mammalian genome modification.
- Existing gene targeting vectors can be improved for efficiency and versatility.
Purpose of the Study:
- To construct and characterize an efficient gene targeting vector, pGT-V1, for mammalian genome engineering.
- To enhance gene knockout efficiency and recipient cell health in transgenic research.
Main Methods:
- Construction of the pGT-V1 vector using plasmid pBS246, incorporating positive (neo, EGFP) and negative (HSV-tk) selection markers.
- Flanking selection markers with LoxP sites for marker removal.
- Inclusion of multiple cloning sequences (MCS-1, MCS-2) with '8 bp cutter' enzyme sites.
- Functional characterization in C2C12 cells.
Main Results:
- The pGT-V1 vector enables instant monitoring of transfection rates via EGFP, improving gene knockout efficiency.
- EGFP marker removal post-knockout is feasible using flow cytometry or immunomagnetic beads, minimizing recipient cell damage.
- '8 bp cutter' sites enhance vector versatility for diverse genetic manipulations.
Conclusions:
- The pGT-V1 plasmid represents an optimized gene targeting vector.
- This vector offers a novel technical approach for transgenic animal research and genome modification.
Related Concept Videos
01:16In-vitro Mutagenesis
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
02:53Conservative Site-specific Recombination and Phase Variation
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...