PiggyBac mediated multiplex gene transfer in mouse embryonic stem cell

Area of Science:

• Molecular Biology
• Genetics
• Stem Cell Biology

Background:

• The PiggyBac system is known for efficient gene transfer.
• Multiplex gene transfer efficiency in mouse embryonic stem cells (ES cells) using PiggyBac remains uncharacterized.
• Development of novel tools is crucial for advanced genetic manipulation in ES cells.

Purpose of the Study:

• To establish an immortalized feeder cell line with multiple antibiotic resistance genes using the PiggyBac system.
• To quantitatively characterize the multiplex transposition efficiency of the PiggyBac system in mouse ES cells.
• To assess the utility of PiggyBac for simultaneous multiplex gene transfer in biomedical research.

Main Methods:

• Utilized the PiggyBac system to introduce four distinct antibiotic resistance genes into SNL 76/7 feeder cells, creating an immortalized cell line.
• Developed five transposons, each containing unique inducible fluorescence proteins and antibiotic resistance genes.
• Quantitatively assessed PiggyBac transposition efficiency in mouse ES cells using these transposons.

Main Results:

• Successfully established a feeder cell line with the most diverse array of antibiotic resistance genes reported to date.
• Demonstrated PiggyBac system efficiency in mouse ES cells, ranging from approximately 2% for single transposon transfer to 0.5% for five transposon transfer.
• Quantified the decrease in efficiency with an increasing number of simultaneously transferred genes.

Conclusions:

• The PiggyBac system is highly efficient for multiplex gene transfer in mouse ES cells.
• The newly developed feeder cell line facilitates simultaneous multiplex gene transfer and targeting experiments.
• This advancement offers researchers expanded options for gene manipulation in biomedical research and development.