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A flow cytometry technique for measuring chromosome-mediated gene transfer
S Vanderbyl1, N MacDonald, G de Jong
1Chromos Molecular Systems, Inc., Burnaby, British Columbia, Canada.
Cytometry
|May 30, 2001
Summary
Researchers developed a rapid screening technique using artificial chromosome expression systems (ACes) to quantify foreign DNA delivery into cells. This method enables efficient optimization of gene transfer technologies for potential gene therapy applications.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Developed a novel screening technique utilizing artificial chromosome expression systems (ACes) for rapid quantification of foreign DNA delivery into cells.
- Delivery efficiency was measured within 24 hours post-transfection using flow cytometry to detect thymidine analogue-labeled ACes.
- The technique is applicable for optimizing ACes and heterologous DNA delivery parameters in cells and tissues.
Purpose of the Study:
- To establish and validate a high-throughput screening method for assessing artificial chromosome (AC) delivery into mammalian cells.
- To compare the efficiency of different transfection reagents (LipofectAMINE 2000 and Superfect) for AC delivery.
- To demonstrate the utility of iododeoxyuridine (IdUrd) labeling for sensitive detection of ACs post-transfection.
Main Methods:
- Chinese hamster ovary (CHO) cells were cultured with iododeoxyuridine (IdUrd) to label artificial chromosomes.
- Purified IdUrd-labeled ACs were transfected into Chinese hamster lung fibroblast (V79-4) and mouse connective tissue (LM(tk-)) cells using LipofectAMINE 2000 and Superfect.
- Transfected cells were analyzed by flow cytometry using an FITC-conjugated anti-bromodeoxyuridine (BrdUrd) antibody to detect AC delivery.
Main Results:
- Intact artificial chromosomes were detected in recipient cells within 2 to 48 hours post-transfection.
- Maximum delivery rates of 20% and 14% were achieved with LipofectAMINE 2000 and Superfect, respectively.
- IdUrd labeling provided superior fluorescence intensity and reduced quenching compared to BrdUrd labeling for flow cytometry analysis.
Conclusions:
- IdUrd-labeled ACs are reliably detectable 24 hours post-transfection, establishing a sensitive, high-throughput screening method.
- This technique facilitates the optimization of various gene transfer technologies, including electroporation and sonoporation, and delivery reagents.
- This study represents a foundational step towards employing artificial chromosomes as nonviral vectors for gene therapy.