What is a DNA delivery system? A DNA delivery system is a method by which exogenous DNA is introduced into an organism. These systems can be divided into two general categories: viral delivery systems and the non viral delivery systems.
The viral delivery systems are the ones that use the viral infection mechanism to deliver genetic material, usually in a viral particle incapable of replicating itself.
On the other hand, the non viral delivery system comprehends a bigger set of strategies:
● Electroporation, which is the introduction of DNA into a cell without a wall using an electric field to form pores in the membrane.
● Microinjection is another mechanism which consists in introducing DNA by pressure into an isobaric system.
● Lipofection as well is a strategy that introduces genetic material by using particles known as liposomes.
● Among others.
DNA Delivery Systems on Eukaryotes
With evidence on induced Pluripotent Stem Cells (iPS cells).
Justification
Cell reprogramming for generation of iPS cells is a technique which uses transduction systems with “reprogrammation factors”, proteins and transcriptions factors involved primarily in the methylation patterns in the genome of a differentiated cell, which are modified in order to produce a cell capable of differentiating again, into different types of cells, but not a whole organism. While this reprogrammation might seem different from the “biohacker” system we propose, it is the same basic principle; the use of a gene/DNA delivery system, in this case, for a mammalian cell; the uses of this biohacker system with this type of DNA delivery system will be discussed, while reprogrammation efficiency cannot be discussed, since it relies on more complex factors, as the complete modification of methylation patterns, the stability of the new methylation patterns and a steady state of other differentiation and growth factors.
Retroviruses.
A common tool, used in both clinical gene therapy and basic research, are retroviruses; their biology is well understood, and they have a high efficiency on both transduction and expression of genes, and can be replication-competent (it has in its genome the essential genes for virion synthesis) and replication defective; thus, the technique is widely used. Nevertheless, the virus genome is large, so it has a limited cloning capacity for multiple genes, which makes a reprogrammation difficult on whole systems or modules on synthetic biology or induction of pluripotent cells.
Even though, in the work of Yamanaka, the first delivery of four reprogrammation factors on rat fibroblasts in order to make iPS was made using a Moloney murine leukemia virus (MMLV) based retrovirus vector, but this system often reactivated the MMLVLTR promoter, causing tumors due to expression of c-Myc factor; when this promoter was removed, they got completely functional iPS cells.
Better results have been reported with other strains of cells, reprogrammation factors or retroviruses.
Lentiviruses.
In contrast with retroviruses, lentiviral systems can infect both dividing and non-dividing cells, whilst retroviral systems can only infect dividing cells. All other features are shared, including retrotranscription and genome integration.
Though lentiviral systems integration is unpredictable, it is less likely that they integrate in oncogenic regions of the genome, causing cancer, than gamma-retroviral systems. Unlike the retroviral systems, all of the lentiviral systems have been designed replication-defective.
Episomal vectors
Most plasmids cannot replicate themselves in a mammalian environment, therefore, they only express transiently. Nevertheless, plasmids like oriP/Epstein-Barr nuclear antigen-1 replicate autonomously as extrachromosomal elements without integration in cells, dividing or non-dividing.
Adenovirus
Adenoviral systems are also non-integrative vectors (except on eggs), can transfect both replicative and non-replicative systems, as well as almost every kind of cell, except for certain lymphoid cells.
Others
In iPS cells, excisable integrated vectors are also used in order to generate iPS transgene free cells, capable of differentiating, but the biohacker system itself does not consider the excision of the transgenes at any moment, it rather focuses on the introduction of new programs in the same cell, as a way of direct “differentiation”, so excisable integrated vectors were not considered.
Animal-bacteria horizontal gene transfer.
Horizontal gene transfer has been described in a wide variety of organisms, mostly prokaryotes; whole genome analyses show that genes are transferred horizontally between closely related taxa, and between bacteria inhabiting the same environment.
Horizontal gene transfer is also found in eukaryotes, and even on superior animals; examples range from the transfer of P elements between Drosophila melanogaster and D. willistoni, the transfer of genes for carotenoid biosynthesis from fungi to pea aphids and the more recently discovered heritable lateral gene transfer in humans and Trypanosoma cruzi.
Although rare, interkingdom gene transfer has also been observed, the Eubacteria Thermotoga maritime has 81 archeal genes, and the well known Eubacteria-Eukaryota transference; Agrobacterium tumefaciens transfers 10-30kbp to plants. In the case of bacteria to animals, it happens through symbiosis, like the symbiotic relationship between Wolbachia with arthropods and filarial nematodes.
Though not a DNA delivery system in itself, the danger of transferring the program to other species, bacteria, animals or plants, has to be considered in function of the organism we are using.
DNA delivery system.
Delivery system
Description
Viability of in-situ application
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Biolistics
DNA coated microparticles (Au/W) are accelerated at high velocities to penetrate membranes or cell walls. (1)
No
Electroporation
High voltage electric pulses permeabilize temporarily the cell membrane, allowing the DNA entry. (2)
No
Bacterial gram+ transformation with glass beads
Bacterial protoplasts are agitated with glass beads in the presence of DNA and PEG. (3)
No
Chemical Methods
Bacterial cells acquire competency with a cold divalent cation solution and after a brief heat shock, DNA is introduced into the cell's interior (4)
No
Phague-based DNA transfer
A phagemid is used to store the DNA of interest, the culture with the cells containing the phagemid is lysed, and the supernatant which holds the phagemid is used to transduced a culture. (5)
Yes
References
Gene transfer. Biolistics delivery systems. BIO-RAD. Retrieved from http://bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5443.pdf
Weavera J., Y. Chizmadzhevb. 1996. Theory of electroporation: A review. Bioelectrochemistry and Bioenergetics 41(2): 135–160.
Rattanachaikunsopon P., P. Phumkhachorn. 2009. Glass bead transformation method for gram-positive bacteria. Braz J Microbiol. 40(4): 923–926.
University of Massachussets Medical School. Transformation of Bacterial Cells. Regional Science Resource Center. Retrieved from https://www.umassmed.edu/uploadedFiles/trans%20bact%20cells(1).pdf
Kittleson J., W. DeLoache, H. Cheng, J. Anderson. 2012. Scalable Plasmid Transfer using Engineered P1-based Phagemids. ACS Synth. Biol. 1, 583−589.
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