Related Experiment Video
Updated: Aug 8, 2026
Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants
Published on: December 16, 2016
Transfer of Liposome-Sequestering Plasmid DNA into Daucus carota Protoplasts
1Institute of Biological Sciences, University of Tsukuba, Sakura-mura, Ibaraki-ken 305, Japan.
Abstract:
Reverse-phase evaporation lipid vesicles (REV) liposomes, consisting of phosphatidyl choline and stearylamine in 1:3 molar ratio, encapsulated approximately 30% of exogenously supplied recombinant DNA vector, pBR322. The DNA sequestered in REV liposomes was highly tolerant to DNase.A two-step procedure was developed, which involves encapsulation of DNA with liposomes using one-tenth phosphate-buffered saline-0.5 molar mannitol, followed by incubation of liposome-DNA with protoplasts in phosphate buffer-0.5 molar mannitol.About 11% of liposome-encapsulated DNA was transferred into protoplasts, whereas 6% uptake was observed in the control. Although some degradation of incorporated DNA occurred inside protoplasts, 50% of the total radioactivity resolved by 0.8% agarose gel was associated with pBR322 forms in 5-hour incubation. After 20-hour incubation, open circular DNA disappeared completely and maintenance of covalently closed circular DNA was confirmed.
Insights
Liposomes effectively encapsulate recombinant DNA, protecting it from degradation. This method enhances DNA transfer into protoplasts, with stable DNA forms maintained long-term.
Area of Science:
- Biotechnology
- Molecular Biology
- Drug Delivery Systems
Background:
- Liposomes are versatile carriers for biomolecules.
- Efficient delivery of recombinant DNA into cells is crucial for genetic applications.
- Protecting DNA from enzymatic degradation is a key challenge.
Purpose of the Study:
- To develop and evaluate a liposome-based system for encapsulating and delivering recombinant DNA (pBR322) into protoplasts.
- To assess the stability and integrity of the encapsulated DNA post-transfer.
Main Methods:
- Reverse-phase evaporation (REV) liposomes were formulated using phosphatidyl choline and stearylamine.
- A two-step procedure was employed for DNA encapsulation and subsequent incubation with protoplasts.
- DNA integrity and transfer efficiency were analyzed using agarose gel electrophoresis and radioactivity assays.
Main Results:
- REV liposomes encapsulated approximately 30% of the pBR322 DNA, exhibiting high DNase tolerance.
- An 11% transfer of liposome-encapsulated DNA into protoplasts was achieved, compared to 6% in controls.
- Covalently closed circular DNA forms were maintained for up to 20 hours post-incubation, despite some degradation.
Conclusions:
- Liposome-mediated delivery offers a promising strategy for introducing recombinant DNA into protoplasts.
- The REV liposome system provides protection against DNA degradation and facilitates stable DNA maintenance within cells.
Related Concept Videos
02:50Transgenic Plants
The first-ever transgenic plant was a tobacco plant developed in 1983 that showed resistance against the tobacco mosaic virus. Since then, many transgenic plants have been developed and commercialized for improving the agricultural, ornamental, and horticultural value of a crop plant. Transgenic...
02:19Export of Mitochondrial and Chloroplast Genes