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The MultiBac Protein Complex Production Platform at the EMBL
Published on: July 11, 2013
Bacterial delivery of large intact genomic-DNA-containing BACs into mammalian cells
Wing Cheung1, George Kotzamanis, Hassan Abdulrazzak
1Division of Natural Sciences, Imperial College London, London, UK.
Abstract:
Efficient delivery of large intact vectors into mammalian cells remains problematical. Here we evaluate delivery by bacterial invasion of two large BACs of more than 150 kb in size into various cells. First, we determined the effect of several drugs on bacterial delivery of a small plasmid into different cell lines. Most drugs tested resulted in a marginal increase of the overall efficiency of delivery in only some cell lines, except the lysosomotropic drug chloroquine, which was found to increase the efficiency of delivery by 6-fold in B16F10 cells. Bacterial invasion was found to be significantly advantageous compared with lipofection in delivering large intact BACs into mouse cells, resulting in 100% of clones containing intact DNA. Furthermore, evaluation of expression of the human hypoxanthine phosphoribosyltransferase (HPRT) gene from its genomic locus, which was present in one of the BACs, showed that single copy integrations of the HPRT-containing BAC had occurred in mouse B16F10 cells and that expression of HPRT from each human copy was 0.33 times as much as from each endogenous mouse copy. These data provide new evidence that bacterial delivery is a convenient and efficient method to transfer large intact therapeutic genes into mammalian cells.
Insights
Bacterial invasion efficiently delivers large intact DNA vectors (BACs) into mammalian cells, outperforming lipofection. This method ensures 100% intact DNA delivery and enables gene expression, offering a new tool for gene therapy.
Area of Science:
- Molecular Biology
- Cell Biology
- Gene Therapy
Background:
- Efficient delivery of large DNA vectors into mammalian cells is a significant challenge in biotechnology and gene therapy.
- Bacterial invasion offers a potential alternative to traditional transfection methods for large DNA constructs.
Purpose of the Study:
- To evaluate the efficiency of bacterial invasion for delivering large intact bacterial artificial chromosomes (BACs) into mammalian cells.
- To compare bacterial invasion with lipofection for delivering large DNA vectors.
- To assess the functionality and expression of a delivered gene from a BAC.
Main Methods:
- Bacterial invasion was used to deliver BACs (over 150 kb) into various mammalian cell lines.
- The effect of chloroquine on bacterial delivery efficiency was investigated.
- Lipofection was used as a comparative delivery method.
- Gene expression analysis of the human hypoxanthine phosphoribosyltransferase (HPRT) gene delivered via BAC was performed.
Main Results:
- Bacterial invasion significantly increased delivery efficiency by 6-fold in B16F10 cells, particularly with chloroquine treatment.
- Bacterial invasion achieved 100% intact DNA delivery of large BACs into mouse cells, superior to lipofection.
- Single-copy integration and functional expression of the HPRT gene from the delivered BAC were observed in mouse cells.
Conclusions:
- Bacterial invasion is a highly efficient and convenient method for delivering large intact DNA vectors into mammalian cells.
- This technique overcomes limitations of current methods for large DNA delivery, showing promise for therapeutic gene transfer.
- The successful expression of a delivered gene demonstrates the potential of bacterial invasion for gene therapy applications.
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