Transfer of DNA into host cells- the various methods
The recombinant vector carrying foreign DNA needs to be transferred into the suitable host cells. Several methods have been developed for introduction of recombinant DNA molecule into host cells.
According to types of vectors and host cells, the methods are adopted. Some of the methods of gene transfer into host cells are briefly discussed below:
1. Transformation :
Introduction of rDNA molecules into a living cell is called transformation. The DNA molecule comes in the contact of cell surface. Then it is taken up by the host cells.
However, in nature the frequency of transformation of many cells (e.g. yeast and mammalian cells) is very less. Secondly, all the time host cells do not undergo transformation. Because they are not prepared for it.
When there develops competence factors in the cells, transformation phenomenon occurs. There are some factors which affect transformation such as concentration of foreign DNA molecules, host's cell density, temperature, etc.
A temperature sock of either low temperature (0-5°C) or high temperature (37- 45°C) is required for transformation. Mandel and Higa (1970) reported that E. coli cells become competent to uptake DNA by treating cells with ice cold CaCl2 and exposing the cells to 42°C for about 90 seconds.
2. Transfection :
Transfection is the transfer of foreign DNA into cultured host cells mediated through chemicals.
The charged chemical substances such as cationic liposomes, calcium phosphate of DEAE dextran are taken and mixed with DNA molecules. The recipient host cells are overlayed by this mixture. Consequently the foreign DNA is taken up by the host cells.
3. Electro poration (Electric Field-mediated Membrane Permeation) :
In electroporation an electric current at high voltage (about 350 V) is applied in a solution containing foreign DNA and fragile host cells. This creates transient microscopic pores in cell membrane of naked protoplasts.
Consequently foreign DNA enters into the protoplast through these pores. The transformed protoplasts are cultured in vitro which regenerate respective cell walls.
4. Microinjection :
In this technique foreign DNA is directly and forcibly injected into the nucleus of animal and plant cells through a glass micropipette containing very fine tip of about 0.5 mm diameter.
It resembles with injection needle. In 1982, for the first time Rubin and Spradling introduced Drosophila gene into P-element and microinjected into embryo. In 1982, R.D. Palmiter and R.L.
It refers to the process of using a glass micropipette to inject a liquid substance at a microscopic or borderline macroscopic level. The target is often a living cell but may also include intercellular space. Microinjection is a simple mechanical process usually involving an inverted microscope with a magnification power of around 200x (though sometimes it is performed using a dissecting stereo microscope at 40-50x or a traditional compound upright microscope at similar power to an inverted model).
For processes such as cellular or pronuclear injection the target cell is positioned under the microscope and two micromanipulators— one holding the pipette and one holding a microcapillary needle usually between 0.5 to 5 µmin diameter (larger if injecting stem cells into an embryo)— are used to penetrate the cell membrane and/or the nuclear envelope. In this way the process can be used to introduce a vector into a single cell. Microinjection can also be used in the cloning of organisms, in the study of cell biology and viruses, and for treating male sub fertility through intracytoplasmic sperm injection
5. Particle Bombardment Gun (Biolistics) :
This technique was developed by Stanford in 1987.
A gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information.
The payload is an elemental particle of a heavy metal coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics.
This device is able to transform almost any type of cell, including plants, and is not limited to genetic material of the nucleus: it can also transform organelles, including plasmids. In this method macroscopic gold or tungusten particles are coated with desired DNA. A plastic micro-carrier containing DNA coated gold/tungusten particles are placed near rupture disc.
The particles are bombarded onto target cells by the bombardment apparatus. Consequently foreign DNA is forcibly delivered into the host cells.
Applications
Gene guns are so far mostly applied for plant cells. However, there is much potential use in animals and humans as well.
Plants
The target of a gene gun is often a callus of undifferentiated plant cells growing on gel medium in a petri dish. After the gold particles have impacted the dish, the gel and callus are largely disrupted. However, some cells were not obliterated in the impact, and have successfully enveloped a DNA coated gold particle, whose DNA eventually migrates to and integrates into a plant chromosome.
Cells from the entire petri dish can be re-collected and selected for successful integration and expression of new DNA using modern biochemical techniques, such as a using a tandem selectable gene and northern blots.
Selected single cells from the callus can be treated with a series of plant hormones, such as auxins and gibberellins, and each may divide and differentiate into the organized, specialized, tissue cells of an entire plant. This capability of total re-generation is called totipotency. The new plant that originated from a successfully shot cell may have new genetic (heritable) traits.
The use of the gene gun may be contrasted with the use of Agrobacterium tumefaciens and its Ti plasmid to insert genetic information into plant cells. See transformation for different methods of transformation in different species.
Humans and animals
Gene guns have also been used to deliver DNA vaccines.
The delivery of plasmids into rat neurons through the use of a gene gun, specifically DRG neurons, is also used as a pharmacological precursor in studying the effects of neurodegenerative diseases such as Alzheimer's Disease.
The gene gun technique is also popularly used in an edible vaccine production technique, where the nano-gold particles coated with plant genetic material under the high vacuum pressurized chamber are transformed into suitable plant tissues.
The gene gun has become a common tool for labeling subsets of cells in cultured tissue. In addition to being able to transfect cells with DNA plasmids coding for fluorescent proteins, the gene gun can be adapted to deliver a wide variety of vital dyes to cells.
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6. Agrobacterium-mediated Gene Transfer :
As discussed earlier that foreign DNA is inserted into Ti-plasmid of Agrobacterium tumifaciens. The recombinant plasmid is transferred into A. tumifaciens cells. The genetically modified A. tumifaciens cells are co-cultured with plant cells in vitro.
Genetically modified A. tumifaciens infects the cultured plant cell and delivers foreign DNA containing plasmid into the infected cell.
A similar technique is also used to transfer foreign gene into root cells by using genetically modified Agrobacterium rhizogenes cells.
Sources:
Content:
https://en.wikipedia.org/wiki/Microinjection
https://en.wikipedia.org/wiki/Gene_gun
Images:
https://www.biontex.com/con_4_6_4/cms/front_content.php?changelang=1&idart=198
https://www.btxonline.com/pages/FAQ.html
https://www.tutorvista.com/content/biology/biology-iii/biotechnology/gene-transfer.php
https://www.nepadbiosafety.net/subjects/biotechnology/plant-transformation-bombardment