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Electroporation, or electropermeabilization, is a microbiology technique in which an electrical field is applied to cells in order to increase the permeability of the cell membrane, allowing chemicals, drugs, electrode arrays or DNA to be introduced into the cell. In microbiology, the process of electroporation is often used to transform bacteria, yeast, or plant protoplasts by introducing new coding DNA. If bacteria and plasmids are mixed together, the plasmids can be transferred into the bacteria after electroporation, though depending on what is being transferred, cell-penetrating peptides or CellSqueeze could also be used. Electroporation works by passing thousands of volts across suspended cells in an electroporation cuvette. Afterwards, the cells have to be handled carefully until they have had a chance to divide, producing new cells that contain reproduced plasmids. This process is approximately ten times more effective in increasing cell membrane's permeability than chemical transformation.
A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.
Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during either transcription or translation and is often used in research. In particular, methods used to silence genes are being increasingly used to produce therapeutics to combat cancer and other diseases, such as infectious diseases and neurodegenerative disorders.
Gene knockdown is an experimental technique by which the expression of one or more of an organism's genes is reduced. The reduction can occur either through genetic modification or by treatment with a reagent such as a short DNA or RNA oligonucleotide that has a sequence complementary to either gene or an mRNA transcript.
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, is a class of double-stranded RNA at first non-coding RNA molecules, typically 20-24 base pairs in length, similar to miRNA, and operating within the RNA interference (RNAi) pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation.
Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term as transformation is also used to refer to progression to a cancerous state (carcinogenesis) in these cells. Transduction is often used to describe virus-mediated gene transfer into eukaryotic cells.
Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA, and it is DNase resistant. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.
In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein to cells. By coating particles of a heavy metal with a gene of interest and firing these micro-projectiles into cells using mechanical force, an integration of desired genetic information can be introduced into desired cells. The technique involved with such micro-projectile delivery of DNA is often referred to as biolistics.
A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.
A short hairpin RNA or small hairpin RNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.
Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.
Lipofectamine or Lipofectamine 2000 is a common transfection reagent, produced and sold by Invitrogen, used in molecular and cellular biology. It is used to increase the transfection efficiency of RNA or plasmid DNA into in vitro cell cultures by lipofection. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the transfection payload, e.g. DNA plasmids.
Magnetofection is a transfection method that uses magnetic fields to concentrate particles containing vectors to target cells in the body. Magnetofection has been adapted to a variety of vectors, including nucleic acids, non-viral transfection systems, and viruses. This method offers advantages such as high transfection efficiency and biocompatibility which are balanced with limitations.
Minicircles are small (~4kb) circular replicons. They occur naturally in some eukaryotic organelle genomes. In the mitochondria-derived kinetoplast of trypanosomes, minicircles encode guide RNAs for RNA editing. In Amphidinium, the chloroplast genome is made of minicircles that encode chloroplast proteins.
Nucleofection is an electroporation-based transfection method which enables transfer of nucleic acids such as DNA and RNA into cells by applying a specific voltage and reagents. Nucleofection, also referred to as nucleofector technology, was invented by the biotechnology company Amaxa. "Nucleofector" and "nucleofection" are trademarks owned by Lonza Cologne AG, part of the Lonza Group.
Dendrosomes are novel vesicular, spherical, supramolecular entities wherein the dendrimer–nucleic acid complex is encapsulated within a lipophilic shell. They possess negligible hemolytic toxicity and higher transfection efficiency, and they are better tolerated in vivo than are dendrimers. The word " Dendrosome" came from the Greek word "Dendron" meaning tree and " some" means vesicles. Thus dendrosomes are vesicular structures composed of dendrimers.
Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by several methods, summarized below. The two major classes of methods are those that use recombinant viruses and those that use naked DNA or DNA complexes.
Ram I. Mahato is a professor and chairman of the Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, United States. He was Professor of Pharmaceutical Sciences and Biomedical Engineering at the University of Tennessee Health Science Center, Memphis. He was Research Assistant Professor at the University of Utah, Senior Scientist at GeneMedicine Inc and a postdoctoral fellow at the University of Southern California in Los Angeles. Washington University in St. Louis, and Kyoto University, Kyoto, Japan. He received a PhD in drug delivery from the University of Strathclyde, UK and BS from China Pharmaceutical University, Nanjing. He is a CRS Fellow, AAPS Fellow, Permanent Member of BTSS/NIH Study section (2009–2013), and ASGCT Scientific Advisor.
Transient expression, more frequently referred to "transient gene expression", is the temporary expression of genes that are expressed for a short time after nucleic acid, most frequently plasmid DNA encoding an expression cassette, has been introduced into eukaryotic cells with a chemical delivery agent like calcium phosphate (CaPi) or polyethyleneimine (PEI). However, unlike "stable expression," the foreign DNA does not fuse with the host cell DNA, resulting in the inevitable loss of the vector after several cell replication cycles. The majority of transient gene expressions are done with cultivated animal cells. The technique is also used in plant cells; however, the transfer of nucleic acids into these cells requires different methods than those with animal cells. In both plants and animals, transient expression should result in a time-limited use of transferred nucleic acids, since any long-term expression would be called "stable expression."
An mRNAvaccine is a type of vaccine that uses a copy of a molecule called messenger RNA (mRNA) to produce an immune response. The vaccine delivers molecules of antigen-encoding mRNA into immune cells, which use the designed mRNA as a blueprint to build foreign protein that would normally be produced by a pathogen or by a cancer cell. These protein molecules stimulate an adaptive immune response that teaches the body to identify and destroy the corresponding pathogen or cancer cells. The mRNA is delivered by a co-formulation of the RNA encapsulated in lipid nanoparticles that protect the RNA strands and help their absorption into the cells.
References
- ↑ "UW biotech spinoff Mirus Bio celebrates two decades in business". news.wisc.edu. Retrieved 2022-03-22.
- ↑ Budker V, Zhang G, Knechtle S, Wolff JA. Naked DNA delivered intraportally expresses efficiently in hepatocytes. Gene Ther. 1996 Jul;3(7):593-8.
- ↑ Budker VG, Zhang G, Danko I, Williams P, Wolff J. The efficient expression of intravascularly delivered DNA in rat muscle. Gene Ther 1998 5: 272–276.
- ↑ Trubetskoy VS, Loomis A, Slattum PM, Hagstrom JE, Budker VG, Wolff JA. Caged DNA does not aggregate in high ionic strength solutions. Bioconjug Chem. 1999 Jul-Aug;10(4):624-8.
- ↑ Lewis DL, Hagstrom JE, Loomis AG, Wolff JA, Herweijer H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat Genet. 32:107, 2002.
- ↑ Trubetskoy VS, Wong SC, Subbotin V, Budker VG, Loomis A, Hagstrom JE, Wolff JA. Recharging cationic DNA complexes with highly charged polyanions for in vitro and in vivo gene delivery. Gene Ther. 2003 Feb;10(3):261-71.
- ↑ Hagstrom JE, Hegge J, Zhang G, Noble M, Budker V, Lewis DL, Herweijer H, Wolff JA. A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs. Mol Ther. 2004 Aug;10(2):386-98.
- ↑ Bates MK, Zhang G, Sebestyen MG, Neal ZC, Wolff JA, Herweijer H. Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA. Biotechniques. 2006 Feb;40(2):199-208.
- ↑ Rozema DB, Lewis DL, Wakefield DH, Wong SC, Klein JJ, Roesch PL, Bertin SL, Reppen TW, Chu Q, Blokhin AV, Hagstrom JE, Wolff JA. Dynamic PolyConjugates for targeted in vivo delivery of siRNA to hepatocytes. Proc Natl Acad Sci USA 7 August 2007: 104(32): 12982–7.
- ↑ Wakefield DH, Klein JJ, Wolff JA, Rozema DB. Membrane activity and transfection ability of amphipathic polycations as a function of alkyl group size. Bioconjug Chem. 2005 Sep-Oct;16(5):1204-8.
- ↑ Wooddell C, Reppen T, Wolff, JA, Herweijer H. Sustained liver-specific transgene expression from the albumin promoter in mice following hydrodynamic plasmid DNA delivery. J Gene Med10:551, 2008.
