Team:UANL Mty-Mexico/project/DNA-Program-Delivery

From 2014.igem.org

(Difference between revisions)
 
(19 intermediate revisions not shown)
Line 23: Line 23:
}
}
-
#title {height: 55px; padding-left: 10px; background-color: #8A0808; font-size:50px; color:white; font-weight:bold; text-decoration:none; text-align:left;}
+
#title {height: 55px; padding-left: 10px; background-color: #0A2A12; font-size:50px; color:white; font-weight:bold; text-decoration:none; text-align:left;}
-
#subtitle {height: 35px; padding-left: 10px; background-color: #DF0101; font-size:30px; color:white; font-weight:bold; text-decoration:none; text-align:left;}
+
#subtitle {height: 35px; padding-left: 10px; background-color: #0B3B24; font-size:30px; color:white; font-weight:bold; text-decoration:none; text-align:left;}
p {padding: 14px;}
p {padding: 14px;}
Line 32: Line 32:
<body>
<body>
 +
 +
<table id="title" width="100%">
<table id="title" width="100%">
Line 56: Line 58:
<td>
<td>
-
<p><a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Description"><font color="blue">Description</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Objective"><font color="blue">Objective</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Explanation"><font color="blue">Explanation</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Bases"><font color="blue">Bases</font></a> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#Conclusion"><font color="blue">Conclusion</font></a><br><br><b>Synthetic Rally</b><br>
+
<p align="center"><div class="Estilo8"> DNA Delivery System </p></div>
-
<br><a name="Description"></a><b>Description</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
+
 
-
<p align="justify">One of our activities for human practices consisted in creating a rally that would teach synthetic biology to students from 9th grade. We wanted to show something that was, in some cases, difficult to explain. The plan was to teach them some basic things about molecular biology that would help them understand each of the bases of the rally, including genes and biobricks, and how they could use these to design a circuit for a project. We decided that each of the bases would represent different parts of this year’s circuit, explaining how each of the parts work. For example, base one represented the Promoter; base two, the Riboswitch, and so on. </p>
+
<p align="justify"><b> What is a DNA delivery system? </b><br> 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.
-
<p><br><a name="Objective"></a><b>Objective</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
+
 
-
<p align="justify">The objective was mainly to help students understand that genetically modified machines work, basically, like a normal machine. We wanted to explain each of the parts that formed our circuit, how each of them worked according to its function, and how every part is necessary for the circuit to work. But all of these are hard to understand when you can’t see the relation between both types of machines. When you tell people who know nothing about genetic engineering that you are building a genetic machine, they have no idea of what to imagine; of how that could work. So we tried to show it to the students in the simplest way. </p>
+
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.
-
<p><br><a name="Explanation"></a><b>Explanation</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
+
 
-
<p align="justify">The first thing we did when arriving to the school, was to organize all of the materials needed for each one of the bases. While we were doing this, three of our team members went to one of the classrooms that the school had lent us, and explained some basic things about DNA and genes to the students. They explained it in a simple manner, just so the students could understand that these were needed to form the biobricks, and these last ones to form a circuit. But that part was explained later on.</p>
+
On the other hand, the non viral delivery system comprehends a bigger set of strategies:<br> </p>
-
<p align="justify">After finishing the presentation, the 40 students from the first classroom went outside, to the first base. We divided them in two groups, and gave the first twenty of them small ribands of different colors, to divide them in teams for them to compete. The other twenty were kept waiting, while some of us asked them questions for a survey about transgenic food.</p>
+
 
-
<p align="justify">We handed out one small card with the drawing of our circuit to each one of the teams from the first group; a card that we would mark, whenever they finished one of the bases, with the points they had earned. The one who came out first place would receive 4 points; second place would receive 3, and third and fourth place would receive 2. We had one person in charge of writing down the points in each card to keep track of who was winning. After explaining all of this, the rally began. </p>
+
<ul>  
-
<p align="justify">The first thing we did at each one of the bases was to identify the name; say if it was the promoter, the terminator, the RBS… and we then gave an explanation of what it was without explaining completely how it worked. At the end of the activity, we would state the relationship between what they had played and the way the actual part of the circuit functioned. Then the one in charged would yell, and they would change bases. When the first group passed to the second base, the group that was kept waiting entered the first base. </p>
+
<li> ● Electroporation, which is the introduction of DNA into a cell without a wall using an electric field to form pores in the membrane. </li>
-
<p><br><a name="Bases"></a><b>Bases</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
+
<li> ● Microinjection is another mechanism which consists in introducing DNA by pressure into an isobaric system. </li>
-
<p align="justify">''BASE 1: PROMOTER'' - The activity was called Streets and Avenues, but the game was changed a little bit. All the teams would be aligned, forming a square, and one person from each team would be standing at each corner. That person would have to find his/her match; someone with the same color inside the square. For example, maybe someone form team Blue, inside the square, would be Yellow. The Yellow person outside would have to find that Blue person before the others found their match, with the path changing just as it does in the common “Streets and Avenues“ game. </p>
+
<li> ● Lipofection as well is a strategy that  introduces genetic material by using particles known as liposomes. </li>
-
<p align="justify">''How does this relate to the promoter?'' The promoter needs to find the one thing that starts it, for the circuit to start too. </p>
+
<li> ● Among others. </li> </ul> <br>
-
<p align="justify">''BASE 2: RIBOSWITCH'' - This activity was called Dragons. Each team had to form a line and hold hands, being really careful not to let go. The person in the very front would be the head, and the last one, with a bandana hanging on their clothes, would be the tail. The objective was that the head would have to take the other team’s bandana, being careful that they didn’t lose theirs. The moment the team lost theirs, they would lose and have to stop right where they were. </p>
+
 
-
<p align="justify">''How does this relate to the Riboswitch?'' The riboswitch turns the circuit on and off, depending on several conditions. In this game, the team was “On“ while they still had their bandana; they were turned “Off“ when they lost it. </p>
+
<p align="justify"><b> DNA Delivery Systems on Eukaryotes </b><br> </p>
-
<p align="justify">''BASE 3: PROTEIN CODING SEQUENCES'' - This base was one of the hardest to fulfill. We designed a “labyrinth“ of conditions that each team would have to pass through. They would have to dress up with objects in some boxes, and depending on what they wore, they would move through the squares. The one who found the perfect combination would win.
+
 
-
</p>
+
<p align="justify"><b> With evidence on induced Pluripotent Stem Cells (iPS cells). </b><br> <br>
-
<p align="justify">''How does this relate to the protein coding sequence?'' Protein coding sequences, as stated in the Registry of Standard Biological Parts, encode the amino acid sequence of one particular protein. In the game there was only one perfect combination, one combination that would make you win; and you had to find it. </p>
+
 
-
<p align="justify">“BASE 4: RBS“ - This activity was somewhat simple, yet fun anyway. We would have two people holding a piece of fabric next to two other people, also holding a piece of fabric, and they would be passing along a small ball, in several different ways, to a small box on the other side of the field. For example, the first time it would be walking and passing the ball however they could. The second time, they would have to throw it as higher as possible, and running. The third time would be running backwards, and the fourth would be jumping on one foot. Only one team got to the fifth time, and they had to do it backwards and jumping on one foot. At the end, the team with the greatest amount of balls inside their box, won.</p>
+
 
-
<p align="justify">''How does this relate to the RBS?'' The RBS is the place where the ribosomes bind and start the process of translation. The small balls represent the ribosome trying to get to the RBS, the box. </p>
+
Justification <br> <br>
-
<p align="justify">“BASE 5: REPORTER“ - The activity was quite simple, and at the same time complicated. We had to build 4 boxes with wires, batteries and a light bulb; only two wires would be connected to the batteries and could turn on the light bulb. There were 20 wires, and the first team who found the combination and turn on the light, would win.</p>
+
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. [12]<br> <br>
-
<p align="justify">''How does this relate to the Reporter?'' The light would turn on whenever the right wires were connected; it acted like a signal. That’s what the reporter does; when the condition is fulfilled, it sends a signal to let you know, just like the light.</p>
+
-
<p align="justify">“BASE 6: TERMINATOR“ - The last activity was just like a game named Doctor, except that we used balloons instead. Each team would have to get in a closed circle, but instead of holding hands directly, they would hold a large balloon. Then they would have to tangle, without letting go of the balloons, as much as possible. The “doctor“ would then have to undo their “knot“, without breaking the circle. The first ones to blow up or let go of their balloon would lose. </p>
+
        Retroviruses.<br> <br>
-
<p align="justify">''How does this relate to the terminator?“ The terminator causes transcription to stop; it sort of “breaks it off“. In the game, “transcription“ stopped whenever the person blow up or let go of the balloon. </p>
+
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. [13]<br>
-
<p><br><a name="Conclusion"></a><b>Conclusion</b> - <a href="https://2013hs.igem.org/Team:CIDEB-UANL_Mexico/HP-SchoolDiffusion-SyntheticRally#"><font color="blue">Return</font></a></p>
+
-
<p align="justify">At the end of the rally, we gave an explanation of the whole circuit; how every part was connected to make it function. We answered questions, we took a picture, and we prized the winner team with a box of cookies. Some of them asked if we had a page on Facebook, and others just thanked us for coming. </p>
+
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. <br>
-
<p align="justify">One of the girls who was helping us with the rally, was not from the team. She was from second semester, and was really interested in iGEM when she found out about it, so she asked if she could come and help. She was of great support; taking pictures and helping us organize everything. At the end it all worked out, even though the day before we were all going crazy because most of the things were missing. It was a really fun experience. </p>
+
 +
Better results have been reported with other strains of cells, reprogrammation factors or retroviruses. <br><br>
 +
 +
        Lentiviruses. <br> <br>
 +
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.<br>
 +
 +
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.<br> <br>
 +
 +
Episomal vectors <br> <br>
 +
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. <br> <br>
 +
 +
Adenovirus <br> <br>
 +
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<.
 +
  <br> <br>
 +
        Others <br> <br>
 +
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. <br> </p>  
 +
 
 +
 
 +
<p align="justify"><b> Animal-bacteria horizontal gene transfer. </b><br><br>
 +
 
 +
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. [15]<br>
 +
 +
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 <i>Trypanosoma cruzi.</i> [16]<br>
 +
 +
Although rare, interkingdom gene transfer has also been observed, the Eubacteria <i>Thermotoga maritime</i> has 81 archeal genes, and the well known Eubacteria-Eukaryota transference; <i>Agrobacterium tumefaciens</i> transfers 10-30 kbp to plants. In the case of bacteria to animals, it happens through symbiosis, like the symbiotic relationship between <i>Wolbachia</i> with arthropods and filarial nematodes. <br>
 +
 
 +
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. <br> <br> </p>
 +
 
 +
<p><b>Animal yeast transfer systems</b><br>Having explained this, there is a delivery system that uses this horizontal gene transfer in an interspecies system, the delivery of DNA and even RNA using recombinant yeast to transduct
 +
mammalian phagocytic cells. Even if it was designed to produce DNA vaccines, this system
 +
could be used as a DNA delivery system for almost any phagocytic organism, more specifically,  
 +
protozoan, amoebas being the classic phagocytic example.</p>
 +
<p>The design of a vector capable of replicating in both amoebas and yeasts might seem complicated, but finding a real life application for the idea is even more complex.[11]</p>
 +
 
 +
<p><b>Non-living delivery systems.</b><br>Justification. <br>
 +
This particular method of DNA delivery is not important for the idea proposed; since the  
 +
organisms need to be in a controlled environment, in order to be transformed with methods
 +
using non-living delivery systems, in particular nanoparticles (e.g. biolistics). Its use as a
 +
transformation method on the field is limited. [9][10]</p>
 +
<p><b>Plant gene transfer systems.</b><br> Justification.<br>
 +
The idea proposed in this project is meant to be employed on the field, and since plants are one
 +
of the most important GMOs released in the environment, it is necessary to review the delivery
 +
systems on plants as well. This, along with bacteria used for bioremediation techniques, would  
 +
cover the vast majority of GMOs. </p>
 +
<p><i>Agrobacterium tumefasciens. </i><br>
 +
<p><i>Agrobacterium tumefasciens</i> is a species of bacteria widely distributed in soil; it is the oldest and
 +
best researched plant transformation method, though we still lack even though we still lack important amounts of knowledge about the basic mechanisms of recognition, attachment, and integration. </p>
 +
 
 +
<p>Nevertheless, our primary concern about using <i>A. tumefasciens</i> on crops is not its
 +
transformation efficiency, but the possibility of disseminating transgenes in the environment.
 +
While it is true that A. tumefasciencs is widely distributed in soil, most isolates do not contain
 +
this plasmid, and the only genes it can transfer are the vir genes; this genes do not represent an
 +
advantage to the host organism, so they do not survive or overcome the rest of the population. </p>
 +
 
 +
<p>However, if we transform the host organism on the field with an <i>A. tumefasciens</i> circuit, it could
 +
also transform other plants, giving them the traits that the circuit could carry, giving them (or not)
 +
an advantage over other organisms. But the problem is the capability of the bacteria to transform other organisms.  
 +
 
 +
<p>We cannot change a mechanism that we do not understand, so making the bacterium host-specific is impossible, since we do not know what proteins are involved in host attachment and recognition.[8]</p>
 +
 
 +
<p>Virus<br>The use of virus to genetically modify an organism is mostly reduced to promoters and other regulatory elements of the virus, since plant virus are not widely studied (except for TMV); but most of the known ones are host-specific.</p>
 +
 
 +
<p>In plants, there’s a very special kind of virus, called “virions”, they are RNA particles with no mRNA activity, whose use as a DNA delivery system is null.</p>
 +
 
 +
<p>Continuing with the virus subject, as in animals, the genetic material of plant virus can be RNA or DNA, never both. Among the RNA virus are the families Tobacovirus (turnip vein-clearing virus, tobacco mosaic virus) and Potexvirus (Alternanthera mosaic virus and potato virus X), and among DNA (which are relevant to us) are the Geminiviridae; small viruses (2.5-3.0 kb per single stranded DNA circle) which replicate within the nucleus, completely depending on host proteins to complete their life cycle.</p>
 +
 
 +
<p>Which means another transformation method, one involving the modification of certain sequences in the Geminivirus, along with the addition of the relevant vir genes from A. tumefasciens or a transposon linked sequence, and our circuit, can be proposed.This way we would have a host-specific delivery system for plants, which could be transfected by a simple rub-inoculation. The system could be replication-competent or replication-deficient, depending on the modified sequences, and integrative or episomal in function of the number of vir genes added.[7]</p>
 +
 
 +
<p>Though not a major set-back, the rub-inoculation method has the disadvantage of limiting the number of specimens that can be transformed at one time; a difficulty that the reprogramator method does not have due to its sprayed inoculation approach.[6]</p>
 +
 
 +
<p><i>Genetic delivery system using specific virus for plants</i><br>A reasonable viral vector option for Maize (Zea mays), one of the most important crops for global agriculture, is the Maize rayadofinomarafivirus (MRFV). These virus, first reported in Zea mays in El Salvador by Ancalmo and Davies (1961), have as its only natural host the Zea mays ssp. mays (maize) and Zea mays ssp. mexicana. It produce chlorotic vein stippling and striping especially in new cultivars, and its transmitted by an insect named Dalbulusmaidis (Cicadellidae). The experimental host range has determined that it have just some few families susceptible, the Gramineae, including three species: Zea mays, Zea mays ssp. mays, and Zea mays ssp. mexicana. Its genome consists of single-stranded RNA with a total genome size of 6.7 kb. Also there are other virus that are good options as gen delivery vectors.</p>
 +
 
 +
 
 +
<p><b>Conclusion</b><br>Virus were chosen as our delivery system,since they are the only specific and safe, both integrative and non integrativesystem, which can be released on the field without fearing any cross contamination; making it the only suitable delivery system for the reprogrammation scheme.</p>
 +
 
 +
 
 +
 
 +
 
 +
<center><img src="https://static.igem.org/mediawiki/2014/e/e5/Dibujo.PNG"></center>
 +
<center><p><i>Table 1</i> General table of DNA delivery systems in bacteria. [1][2][3][4][5]</center>
 +
<p><b>Reference</b>
 +
<li>1. http://bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5443.pdf</li>
 +
<li>2. Weavera J., Y. Chizmadzhevb. 1996. Theory of electroporation: A review. Bioelectrochemistry and Bioenergetics 41(2): 135–160.</li>
 +
<li>3. Rattanachaikunsopon P., P. Phumkhachorn. 2009. Glass bead transformation method for gram-positive bacteria. Braz J Microbiol. 40(4): 923–926.</li>
 +
<li>4. 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</li>
 +
<li>5. Kittleson J., W. DeLoache, H. Cheng, J. Anderson.  2012. Scalable Plasmid Transfer using Engineered P1-based Phagemids. ACS Synth. Biol. 1, 583−589.</li>
 +
<li>6. Scholthof HB.”Rapid delivery of foreign genes into plants by direct rub-inoculation with intact plasmid DNA of a tomato bushy stunt virus gene vector” (1999) Journal of Virology. 73(9): 7823-7829 PMCID: PMC104311</li>
 +
<li>7. Pitzschke A, Hirt H. “New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation” (2010) The EMBO Journal 29: 1021-1032</li>
 +
<li>8. Krenz B, Jeske H, Kleinow T. “The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route” (2012) Frontiers in plant science. ¿? Volume 3, Article 291. doi: 10.3389/fpls.2012.00291</li>
 +
<li>9. Kim T, Lee M, Kim SW “A guanidinylatedbioreducible polymer with high nuclear localization ability for gene delivery systems”(2012) Biomaterials. 31(7): 1798-1816 doi:10.1016/j.biomaterials.2009.19.034</li>
 +
<li>10. Zu Y, Huang S, Liao WC, Lu Y, Wang S. “Gold nanoparticles enhanced electroporation form mammalian cell transfection” (2014) J Biomed Nanotech. 10(6):982-992</li>
 +
<li>11. Walch B, Breinig T, Schmitt MJ, Breinig F. “Delivery of functional DNA and messenger RNA to mammalian phagocytic cells by recombinant yeast” (2012) Gene Therapy 19(3):237-245</li>
 +
<li>12. Shao L, Wu Ws “Gene-delivery systems for iPS cell generation” (2010) Expert OpinBiolTher. 10(2):231-242 doi:10.1517/14712590903455989</li>
 +
<li>13. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S “Generation of mouse induced pluripotent stem cells without viral vectors” (2008) Science 322: 949-952 doi: 10.1126/science.1164270</li>
 +
<li>14. Hotopp JCD “Horizontal gene transfer between bacteria and animals” (2011) Trends on genetics 27(4):157-163 doi:10.1016/j.tig.2011.01.005.</li>
 +
<li>15. Beiko RG, Harlow TJ, Ragan MA “Highways of gene sharing in prokaryotes” (2005) PNAS 102(40):14332-14337</li>
 +
 
 +
</ol>
</td>
</td>
<td style="background-color: #FFFFFF;">
<td style="background-color: #FFFFFF;">
-
<img src="https://static.igem.org/mediawiki/2013hs/d/d3/D9b5b18226b32476e3e16622aae4da9e.jpg" height="300px" />
 
</td>
</td>
</tr>
</tr>
Line 96: Line 194:
</html>
</html>
 +
{{:Team:UANL_Mty-Mexico/footer}}

Latest revision as of 03:53, 18 October 2014

Project
DNA/Program Delivery

DNA Delivery System

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. [12]

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. [13]
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. [15]
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. [16]
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 10-30 kbp 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.

Animal yeast transfer systems
Having explained this, there is a delivery system that uses this horizontal gene transfer in an interspecies system, the delivery of DNA and even RNA using recombinant yeast to transduct mammalian phagocytic cells. Even if it was designed to produce DNA vaccines, this system could be used as a DNA delivery system for almost any phagocytic organism, more specifically, protozoan, amoebas being the classic phagocytic example.

The design of a vector capable of replicating in both amoebas and yeasts might seem complicated, but finding a real life application for the idea is even more complex.[11]

Non-living delivery systems.
Justification.
This particular method of DNA delivery is not important for the idea proposed; since the organisms need to be in a controlled environment, in order to be transformed with methods using non-living delivery systems, in particular nanoparticles (e.g. biolistics). Its use as a transformation method on the field is limited. [9][10]

Plant gene transfer systems.
Justification.
The idea proposed in this project is meant to be employed on the field, and since plants are one of the most important GMOs released in the environment, it is necessary to review the delivery systems on plants as well. This, along with bacteria used for bioremediation techniques, would cover the vast majority of GMOs.

Agrobacterium tumefasciens.

Agrobacterium tumefasciens is a species of bacteria widely distributed in soil; it is the oldest and best researched plant transformation method, though we still lack even though we still lack important amounts of knowledge about the basic mechanisms of recognition, attachment, and integration.

Nevertheless, our primary concern about using A. tumefasciens on crops is not its transformation efficiency, but the possibility of disseminating transgenes in the environment. While it is true that A. tumefasciencs is widely distributed in soil, most isolates do not contain this plasmid, and the only genes it can transfer are the vir genes; this genes do not represent an advantage to the host organism, so they do not survive or overcome the rest of the population.

However, if we transform the host organism on the field with an A. tumefasciens circuit, it could also transform other plants, giving them the traits that the circuit could carry, giving them (or not) an advantage over other organisms. But the problem is the capability of the bacteria to transform other organisms.

We cannot change a mechanism that we do not understand, so making the bacterium host-specific is impossible, since we do not know what proteins are involved in host attachment and recognition.[8]

Virus
The use of virus to genetically modify an organism is mostly reduced to promoters and other regulatory elements of the virus, since plant virus are not widely studied (except for TMV); but most of the known ones are host-specific.

In plants, there’s a very special kind of virus, called “virions”, they are RNA particles with no mRNA activity, whose use as a DNA delivery system is null.

Continuing with the virus subject, as in animals, the genetic material of plant virus can be RNA or DNA, never both. Among the RNA virus are the families Tobacovirus (turnip vein-clearing virus, tobacco mosaic virus) and Potexvirus (Alternanthera mosaic virus and potato virus X), and among DNA (which are relevant to us) are the Geminiviridae; small viruses (2.5-3.0 kb per single stranded DNA circle) which replicate within the nucleus, completely depending on host proteins to complete their life cycle.

Which means another transformation method, one involving the modification of certain sequences in the Geminivirus, along with the addition of the relevant vir genes from A. tumefasciens or a transposon linked sequence, and our circuit, can be proposed.This way we would have a host-specific delivery system for plants, which could be transfected by a simple rub-inoculation. The system could be replication-competent or replication-deficient, depending on the modified sequences, and integrative or episomal in function of the number of vir genes added.[7]

Though not a major set-back, the rub-inoculation method has the disadvantage of limiting the number of specimens that can be transformed at one time; a difficulty that the reprogramator method does not have due to its sprayed inoculation approach.[6]

Genetic delivery system using specific virus for plants
A reasonable viral vector option for Maize (Zea mays), one of the most important crops for global agriculture, is the Maize rayadofinomarafivirus (MRFV). These virus, first reported in Zea mays in El Salvador by Ancalmo and Davies (1961), have as its only natural host the Zea mays ssp. mays (maize) and Zea mays ssp. mexicana. It produce chlorotic vein stippling and striping especially in new cultivars, and its transmitted by an insect named Dalbulusmaidis (Cicadellidae). The experimental host range has determined that it have just some few families susceptible, the Gramineae, including three species: Zea mays, Zea mays ssp. mays, and Zea mays ssp. mexicana. Its genome consists of single-stranded RNA with a total genome size of 6.7 kb. Also there are other virus that are good options as gen delivery vectors.

Conclusion
Virus were chosen as our delivery system,since they are the only specific and safe, both integrative and non integrativesystem, which can be released on the field without fearing any cross contamination; making it the only suitable delivery system for the reprogrammation scheme.

Table 1 General table of DNA delivery systems in bacteria. [1][2][3][4][5]

Reference

  • 1. http://bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_5443.pdf
  • 2. Weavera J., Y. Chizmadzhevb. 1996. Theory of electroporation: A review. Bioelectrochemistry and Bioenergetics 41(2): 135–160.
  • 3. Rattanachaikunsopon P., P. Phumkhachorn. 2009. Glass bead transformation method for gram-positive bacteria. Braz J Microbiol. 40(4): 923–926.
  • 4. 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
  • 5. Kittleson J., W. DeLoache, H. Cheng, J. Anderson. 2012. Scalable Plasmid Transfer using Engineered P1-based Phagemids. ACS Synth. Biol. 1, 583−589.
  • 6. Scholthof HB.”Rapid delivery of foreign genes into plants by direct rub-inoculation with intact plasmid DNA of a tomato bushy stunt virus gene vector” (1999) Journal of Virology. 73(9): 7823-7829 PMCID: PMC104311
  • 7. Pitzschke A, Hirt H. “New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation” (2010) The EMBO Journal 29: 1021-1032
  • 8. Krenz B, Jeske H, Kleinow T. “The induction of stromule formation by a plant DNA-virus in epidermal leaf tissues suggests a novel intra- and intercellular macromolecular trafficking route” (2012) Frontiers in plant science. ¿? Volume 3, Article 291. doi: 10.3389/fpls.2012.00291
  • 9. Kim T, Lee M, Kim SW “A guanidinylatedbioreducible polymer with high nuclear localization ability for gene delivery systems”(2012) Biomaterials. 31(7): 1798-1816 doi:10.1016/j.biomaterials.2009.19.034
  • 10. Zu Y, Huang S, Liao WC, Lu Y, Wang S. “Gold nanoparticles enhanced electroporation form mammalian cell transfection” (2014) J Biomed Nanotech. 10(6):982-992
  • 11. Walch B, Breinig T, Schmitt MJ, Breinig F. “Delivery of functional DNA and messenger RNA to mammalian phagocytic cells by recombinant yeast” (2012) Gene Therapy 19(3):237-245
  • 12. Shao L, Wu Ws “Gene-delivery systems for iPS cell generation” (2010) Expert OpinBiolTher. 10(2):231-242 doi:10.1517/14712590903455989
  • 13. Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S “Generation of mouse induced pluripotent stem cells without viral vectors” (2008) Science 322: 949-952 doi: 10.1126/science.1164270
  • 14. Hotopp JCD “Horizontal gene transfer between bacteria and animals” (2011) Trends on genetics 27(4):157-163 doi:10.1016/j.tig.2011.01.005.
  • 15. Beiko RG, Harlow TJ, Ragan MA “Highways of gene sharing in prokaryotes” (2005) PNAS 102(40):14332-14337
  • Thank you for visiting our wiki. Follow us in:
    facebooktwitter