DNA modification and functional delivery into human cells using Escherichia coli DH10B
Reference:
Narayanan, K. & Warburton, P.E. (2003). DNA modification and functional delivery into human cells using Escherichia coli DH10B. Nucleic Acids Res. 31, e51.
Introduction:
The human genome, in its near entirety, is available via cloned BAC (bacterial artificial chromosome) libraries, which aid in the study of gene expression and other chromosomal functions. However, BACs are difficult to both manipulate and transfer into mammalian cells, due to their large size (contain up to 350 kb of genomic DNA) and the fragile chromosome structures that they form (e.g. centromeres).
Using a recombination-deficient strain of E. coli, such as DH10B, provides greater stability to propagate the BACs, although manipulating the clone then becomes more difficult. In response, a recent method has been developed to induce transient recombination capability to DH10B by introducing them with recombination genes facilitating Gateway Entry technology (GET) while under the regulation of an arabinose-induced promoter.
Additionally, improvements of DNA delivery into mammalian cells are also needed to limit the mechanical degradation that DNA can undergo before reaching its target. Several methods of DNA delivery have been studied, including the expression of the Yersinia pseudotuberculosis invasin gene in E.coli, which allows the bacteria to bind to mammalian integrin receptors and become internalized into the primary vesicles of the mammalian cell.
Method:
This paper sets out to combine GET recombination and bacterial invasion of mammalian cells to develop a novel method for modifying, propagating, and delivering genomic BACs using E.coli DH10B. This method was tested through invasion of HeLa cells, and the delivery of DNA was assessed through green fluorescent protein (GFP) and neomycin expression.
Figures:
Invasive DH10B were cultured overnight on a monolayer culture of HeLa cells at the desired MOI, the ratio of DH10B to HeLa cells. A-E (left) photomicrograph of GFP fluorescence; (center) phase contrast microscopy showing same field of cells; (right) FACS analysis of cells sorted for GFP (x-axis) and auto-fluorescence (y-axis); cells in lower right quadrant represent GFP-positive cells (F, % GFP-expressing cells). (A) MOI = 1000, F = 3.17. (B) MOI = 2000, F = 12.08. (C) MOI = 4000, F = 12.12. (D) Non-invasive E.coli, MOI = 2000, F = 1.67. (E) HeLa colony stably expressing GFP. F = 62.5% after G418 selection for 32 days.
Invasive E.coli is internalized and delivers plasmid and a 200 kb BAC clone into HeLa cells. (A) Internalization of the E. coli DH10B upon exposure to HeLa cells for various time points. Mean values are calculated from two independent experiments. (B) Percentage of GFP expression in HeLa cells by invading E.coli harboring either pEGFP-N2 or hgUroS-1 BAC. Mean values are calculated from three independent experiments. An MOI of 4000 using uninvasive bacteria is shown in leftmost histograms.
Commentary:
This paper presents a novel and innovative method for overcoming some of the limitations of working with cloned BAC libraries. The authors propose that this technique will improve the study of gene expression regulation and even has potential to revolutionize gene therapy. However, before this technique can be used in a medical context, its yield must be significantly improved, as well as its ability to target a specific subset of cells. Currently, at the highest levels of MOI tested, the overnight transfection efficiency was only ~10%. This can present certain dangers, for instance, if only 10% of cancer cells can be eliminated, a bias may be introduced for propagating more malignant tumor cells that are resistant to bacterial invasion. Furthermore, the invasion mechanism used in this paper targets integrin receptors, which are found on most benign mammalian cells. To ensure that therapy is delivered only to diseased cells, the targeting mechanism must be made more specific, or additional mechanisms regulating therapy delivery must be incorporated.
7 comments:
Good analysis of the data but further elaboration on the methods would have made the paper a bit more clear and maybe a little more on the background as well.
Did the researchers study the effects of bacterial incorporation into the HeLa cells? If the bacteria are being incorporated into the HeLa cells, how do they release the BACs? (self-lysis? transfer?) How do the HeLa cells react to bacterial integration? This technique is very interesting and novel and I hope they pursue the topic further with regards to effects on the mammalian cells.
It’s quite interesting how effective this procedure appears to be relative to other methods of transfecting cells with DNA. Although its use as a cancer treatment is obviously limited, it has potential in research as it seems a lot more effective than heat shock, viral vectors, or plasmid transfection. It currently seems unlikely to reach the clinical stage, as introducing infectious bacteria into patients is almost certainly going to be rejected by the FDA.
The greatest strength of this paper is that it combines two existing technologies in a way that is easy to understand and reproduce. However, before this method is adopted by other researchers, the effects of the bacterial invasion on HeLa cells needs to be studied in more detail. The authors find that with longer invasions, the viability of the HeLa cells goes way down, so exposing these cells to invasive bacteria may have unintended effects.
With regards to gene therapy, this technique is still a long way from being used in a clinical setting, but the main goal of the study was to improve current transfection methods and to aid other researchers.
@sharp903
The methods proposed by this paper integrates two well-established techniques, GET recombination and cell invasion via integrin binding. Also, further details concerning the assays performed in this study were summarized in the captions of the figures.
My paper summary touched upon nearly all points that were mentioned in the original introduction of the paper. If you would like more background information on this area of research, I recommend reading some review articles on the subject.
@Jeni
Did the researchers study the effects of bacterial incorporation into the HeLa cells?
Well in this study, bacterial incorporation would produce GFP expression in the HeLa cells. But if you're wondering whether bacterial invasion produces effects on cells in general, it would probably lead to the same response that meets any other bacterial infection. And so, if any invading bacteria were to escape from its entry vesicle, the cell would probably recruit phagocytes to contain the bacteria and kill it.
How do they release the BACs?
The paper is not very clear on this release mechanism. However, it does mention that this strain of E.coli has had a gene knocked out, causing cell wall deficiency. I don't think this allows the BACs to directly seep out of the bacteria. But, I think the inability to synthesize a cell wall leads to self-lysis when the bacteria attempts to replicate, causing its plasmids to spill out and get incorporated by the HeLa cell.
How do the HeLa cells react to bacteria integration?
Pretty well in my opinion, probably due to the method of entry of the bacteria. The bacteria bind to integrin receptors of the HeLa cells, causing the E.coli to be internalized within primary vesicles, and remain sequestered from the rest of the cell. Also, due to the cell wall deficiency, these bacteria cannot reproduce and hijack resources needed to sustain the host cell, and so HeLa viability should not be affected to much by its bacterial guests.
This technique is very interesting and novel and I hope they pursue the topic further with regards to effects on the mammalian cells.
I agree!
@Charles
Good point. I think there still remains a lot of untapped potential for this novel approach to impact research. This bacteria is not infectious, and so I don't think that would be the reason for FDA rejection. But gene therapy, which is what I see this method best applied to, has a poor track record in studies, as it can often induce cancer if integrated into an oncogene. I imagine that would be the main issue to tackle before seeking FDA approval.
@Jay
Yes, the inverse relationship between HeLa cell viability and bacterial incubation time is quite interesting. But I actually believe this is a strength for pursuing the bacteria as a therapy, primarily for anti-cancer treatment. The bacteria could be introduced to the tumor site in order to infect and kill cancer cells. Other benign cells in the vicinity would be protected by the body's immune cells which quickly phagocytose and destroy the bacteria. Since immune cells are much less able to penetrate the tumor environment, fewer bacteria would be eliminated from those regions, creating longer incubation time of the bacteria with tumor cells, which this study showed reduces the cell viability of HeLa, a form of cancer cells.
Good analysis of the paper. I have two questions: 1. What is the exact application of delivering DNA into mammalian cells? and 2. What immunological issues are discussed in association with using DH10B to invade mammalian cells?
This is really interesting as a foundational technology for genetic engineering in mammalian cells via gene delivery. Regarding the second figure, I was wondering about the differences between the two plasmids used (pEGFP-N2 and hgUroS-1 BAC). How did these differences result in the observed differences in GFP expression shown in the graph?
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