Multivalent Retroviral Vectors

[Vmedvil 28]

Rabies/HIV Chimeric Retroviral Vector Gene Map

 

Goes up to MV-5 , Five MV-2 Vectors, Five MV 1 to 5 Vectors.

 

Sample (Nerve/T-cell) Targetting

 

 

 

MV-5 (Nerve,T-Cell,Liver,Lung,Skin) Targetting Retroviral Vector Gene Map

 

Edited May 23, 2018 by Vmedvil

[VictorMedvil 307]

MV-x Hybrid Retroviral Vector design using multiple virus' or any virus' targetting glycoproteins with a C Terminal attachment to target any type of cell specifically. The Above Viral Vectors from MV-1 to MV-5 are examples of this Viral Vector construction method, which the method of creation is Gene Synthesis using oligonucleotide synthesis (cDNA) in the method of Synthetic Viral Synthesis.

 

Edited July 5, 2018 by VictorMedvil

[VictorMedvil 307]

Methodology of Construction Synthetic Viral Synthesis with Pictures = http://www.sciencemadness.org/talk/viewthread.php?tid=76588

 

Step I:Custom Oligonucleotide Synthesis

Materials
•Commerical Nucleic Acid Synthesizer
•Solution of the four DNA phosphoramidite monomers (bases)
•All the 5’-hydroxyl groups must be blocked with a DMT group for all four bases
•All phosphorus linkages must be blocked with a cyanoethyl group.
•Blocking solutions
•Reaction chamber and a type of solid support such as controlled pore glass
•The solid support should be prepared with the desired first base already attached via an ester
linkage at the 3’-hydroxyl end.
•Dichloroacetic acid or trichloroacetic acid
•Tetrazole
•Acetic anhydride and N-methylimidazole
•Dilute iodine in a water/pyridine/tetrahydrofuran solution
•Concentrated ammonia hydroxide.
•Materials for one desalting method

Process

Step A: De-blocking
The first base, which is attached to the solid support, is at first inactive because all the active sites
have been blocked or protected. To add the next base, the DMT group protecting the 5'-hydroxyl
group must be removed. This is done by adding a base, either dichloroacetic acid (DCA) or
trichloroacetic acid in dichloromethane (DCM), to the reaction column. The 5’-hydroxyl group is now the only reactive group on the base monomer. This ensures that the
addition of the next base will only bind to that site. The reaction column is then washed to remove
any extra acid and by-products.

Step B: Base Condensation
The next base monomer cannot be added until it has been activated. This is achieved by adding
tetrazole to the base. Tetrazole cleaves off one of the groups protecting the phosphorus linkage.
This base is then added to the reaction column. The active 5’-hydroxyl group of the preceeding
base and the newly activated phosphorus bind to loosely join the two bases together. This forms
an unstable phosphite linkage. The reaction column is then washed to remove any extra
tetrazole, unbound base and by-products.

Step C: Capping
When the activated base is added to the reaction column some does not bind to the active 5’-
hydroxyl site of the previous base. If this group is left unreacted in a step it is possible for it to
react in later additions of different bases. This would result in an oligonucleotide with a deletion.
To prevent this from occurring, the unbound, active 5’-hydroxyl group is capped with a protective
group which subsequently prohibits that strand from growing again. This is done by adding acetic
anhydride and N-methylimidazole to the reaction column. These compounds only react with the
5’-hydroxyl group. The base is capped by undergoing acetylation. The reaction column is then
washed to remove any extra acetic anhydride or N-methylimidazole.

Step D: Oxidation
In step 2 the next desired base was added to the previous base, which resulted in a unstable
phosphite linkage. To stabalize this linkage a solution of dilute iodine in water, pyridine, and
tetrahydrofuran is added to the reaction column. The unstable phosphite linkage is oxidized to
form a much more stable phosphate linkage.

Repeat as need based on length desired between 1 and 10,000 times.

Final Product: DNA Chains from 1 to 10,000 base pairs in length.

 

 

 

 

Step II:Molecular Cloning: Polymerase Chain Reaction(PCR)

Materials
-Thermal Cycler
-Taq polymerase
-Generated DNA Fragments

Process

A. Denaturation - the DNA is heated usually to 95C to render it single-stranded

B. Annealing - the two primers bind the appropriate complementary strand; the temperature for
this step varies depending on the of size of the primer and its homology to the target DNA

C. Primer Extension - DNA polymerase extends the primer by its polymerase activity; this is
done at a temperature optimal for the particular polymerase that is used; currently the most
popular enzyme for this step is Taq polymerase, the DNA polymerase from the thermophilic
("heat-loving) bacteria Thermus aquaticus; the extension is performed at 72C. These steps are
repeated from 28-35 times. Since the reaction is essentially exponential and since each cycle is
about 5 minutes, a large quantity of DNA can be produced for analysis in as little as several
hours.

Final Product: Exponential DNA cloning

 

 

Step III:Mass Ligation Reaction or Standard Ligation Reaction

Materials
•Two or more fragments of DNA that have either blunt or compatible cohesive ("sticky") ends.
•A buffer which contains ATP. The buffer is usually provided or prepared as a 10X concentrate
which, after dilution, yields an ATP concentration of roughly 0.25 to 1 mM. Most restriction
enzyme buffers will work if supplemented with ATP.
•T4 DNA ligase. A typical reaction for inserting a fragment into a plasmid vector (subcloning)
would utilize about 0.01 (sticky ends) to 1 (blunt ends) units of ligase.

Process
The optimal incubation temperature for T4 DNA ligase is 16C and when very high efficiency
ligation is desired (e.g. making libraries) this temperature is recommended. However, ligase is
active at a broad range of temperatures, and for routine purposes such as subcloning,
convenience often dictates incubation time and temperature - ligations performed at 4C overnight
or at room temperature for 30 minutes to a couple of hours usually work well.


Final Product: Every possible recombination or a Single Recombination

Step IV:Activation and Reproduction : Plasmid Vector
(sub-cloning)

 

 

Materials
-Anything with ribosomes
-NotI Ligase

Process
Same as step III


Final Product: Viral Vector

Edited July 5, 2018 by VictorMedvil

[VictorMedvil 307]

My Science Madness Ebook on Viral Vector Engineering and Bacterial Plasmids along with Biochemistry. This is a series of posts about the subject of Biological Nanorobotics constructed into a Ebook.

 

The Elements of Creation a Guide on Bio-Nanorobotics

 

Introduction: Protoplast Hybridization and Bacterial Plasmid Construction (Bacterial/Cellular Nanomachines)

http://www.sciencemadness.org/talk/viewthread.php?tid=76592

 

Part I: Synthetic Viral Synthesis

http://www.sciencemadness.org/talk/viewthread.php?tid=76588

 

Part 2: Biochemical Construction of a Vector (Viral Nanomachines)

http://www.sciencemadness.org/talk/viewthread.php?tid=76631

 

Part 3: How to make Cats glow in the Dark

http://www.sciencemadness.org/talk/viewthread.php?tid=76633

 

Part 4: Luciferin Metabolism

http://www.sciencemadness.org/talk/viewthread.php?tid=76739

 

Part 5:  Leukemia Cure "Living Drug"

http://www.sciencemadness.org/talk/viewthread.php?tid=76663

 

Part 6: Choosing the Right Vector for the Right Job

http://www.sciencemadness.org/talk/viewthread.php?tid=76775

 

Part 7: Skin cells to Stem Cells

http://www.sciencemadness.org/talk/viewthread.php?tid=76662

 

Part 8: Energy to Protein Biochemistry

http://www.sciencemadness.org/talk/viewthread.php?tid=76725

 

Part 9: Hybridization of Viral Vectors MV-x Strains

http://www.sciencemadness.org/talk/viewthread.php?tid=76935

 

Part 10: Iron Binding Integrase 

http://www.sciencemadness.org/talk/viewthread.php?tid=79041

 

Ending Part 11: Promise and Peril of Molecular Nanotechnology

http://www.sciencemadness.org/talk/viewthread.php?tid=76976

 

 

 

Author: Vmedvil

Edited July 5, 2018 by VictorMedvil

[VictorMedvil 307]

Additional Writings on Hostile Bio-Nanorobotics and Biochemistry

 

The Harvester Project(Black Virology)

https://www.scienceforums.net/topic/112370-harvester-projectsynthetic-black-virology/

 

Does this look Unstable(Organic Explosive)

http://www.sciencemadness.org/talk/viewthread.php?tid=78987

 

Why not to use Harmful Bio-Nanorobotics and why you will fail (Warning against Biological Weapons creation)

http://www.sciencemadness.org/talk/viewthread.php?tid=79707

 

The Academician's private residences shall remain off-limits to the Genetic Inspectors. We possess no retroviral capability, we are not researching retroviral engineering, and we shall not allow this Council to violate faction privileges in the name of this ridiculous witch hunt!

 

Edited July 30, 2018 by VictorMedvil

[VictorMedvil 307]

There is a problem with standard Retroviral Delivery the maximum amount of RNA that can be contained within the virus is limited to 10,000 bp by the caspid size.

 

 

 

The Amount of DNA stored within the HIV-1 ENV can be increased by using a different capsid to contain the DNA, while the RNA length of HIV-1 is around 10,000 bp the length of other viruses is longer like the Chickenpox Virus, has a much longer genome which means that the capsid is larger or is able to contain more Base Pairs around 100,000 bp which is encoded by VP26, which HIV-1's Capsid Gene is P24, so P24 can be replaced by VP26 giving the Vector a carrying capacity of 100,000 base pairs.

 

 

 

Which now the Vector gets a new property of extended carrying capacity making the new gene map and a new name EMV-x for "Extended Multivalent Vector"

Edited March 16, 2020 by VictorMedvil

[VictorMedvil 307]

The N-Targetting(Targets all human cells) and Extremophillic(Wider Temperature Survival Range) Version of the "Extended Retroviral Vector"

 

 

Gene Map

Edited July 5, 2020 by VictorMedvil

[VictorMedvil 307]

These work and have been tested, link = https://www.thelancet.com/journals/lanhiv/article/PIIS2352-3018(19)30262-0/fulltext

Edited March 16, 2020 by VictorMedvil