Transgenesis

1.    Introduction

Transgenesis is the movement or insertion of a gene from one organism to another organism. The main aim of transgenesis is to develop a transgenic organism. Transgenic organisms have a foreign gene or DNA due to which they exhibit a new trait or character. The whole procedure can be done through physical or biological methods. Following steps are generally involved in transgenesis:

1.1.    Identification of gene of interest

The first step is to identify a gene of interest which is coding for a desirable trait. There are many techniques to identify a gene of interest e.g. Gene chip technology.

1.2.    Isolation of the gene

• The targeted gene must be isolated from an organism. It can be done using chemical treatments.
• First of all, DNA is cut by the restriction enzyme.
• The fragments are then separated according to their size using gel electrophoresis.
• The fragment containing a gene of interest can be identified by a DNA probe or primer.
• Then it is cloned using PCR.

1.3.    Transformation

A vector is then used to transfer the targeted gene of interest to an organism.

2.    Methods of transgenesis in various cells and applications

Following are various methods of transgenesis in cells:

2.1.    Physical methods

In the physical methods of gene transmission, various optimized parameters are considered. However, it doesn’t deal with the optimal cell survival and maximal recovery of cell gene lines. But these parameters affect the effective momentum, effective transfer, the amount of useful DNA a gene carries in a particular cell and the number of particles that impact the tissue. Various genes such as B Glucorinidase ensures the optimization of transfer of genes to other particular cells. Biological markers such as green fluorescent protein and luciferase permit the identification of transformed cells soon after the bombardment of genes and provide a suitable method of rapid transformation of genes to other bodies to carry useful purposes.

    2.1.1.    Biostatic gene gun

This is also known as particle bombardment. This was first developed and particularly practiced on plants in the 1980s. it involves shooting a piece of DNA in recipient plant tissues. These plant tissues are been transferred to a particular medium and bombarded with 0.5 mm gold or tungsten filament gun with DNA coated compartments with compressed helium and kept at 30 degrees of Celsius to allow the growth of transformants. In plant cells, DNA is inserted into cells under suitable conditions, so the only drawback is tissue damage and low transformation rate. But this is applicable to both monocotyledons and dicotyledons. To avoid this, gene gun is an effective method for gene transformation to produce transgenic plants. Through this maze, soybeans, wheat, barley is being produced by this method. Chlamydomonas can be transformed into higher genes by bombardment method. Gene gun has various advantages, through these higher rates of genes can be transformed and this is much more effective than other methods. A lot of tissues and particles can be targeted through gene gun and. It also enhances cell survival but there is still some chances of the gene or cell damage. In bacteria, the first gene gun was used on yeast and demonstrated to increase the rate of transformation in plants. In yeast, they found it more effective as no cell wall was more receptive to accept new genes. Through these various variety of useful types of transgenic yeasts are produced.

2.1.2.    Electroporation

The electric field generates holes in the plasma membrane thus allowing the genetic material to get into the cells. But this technique has various drawbacks as creating holes in the plasma membrane can cause the cells to get damaged or leakage of important cellular components out of the cells. So, the survival rate in this type of method is quite low (25-50%).so it is regarded not as an effective method for gene transformation as it can result in the death of the tissues. Plus, electricity or electrical shocks can also alter the genetic makeup of the cells. In plants and bacteria: as compared to animals, plants and bacteria can endure this method as they have a protective covering out of them, so the rate of tissue damage is relatively lesser in it. In animals: in animals, it can be effective or not as animals have only one layered protective covering that allows them to be more vulnerable to electrical damage. But still, in a lot of cases, it is effective.

2.2.    Biological methods

Gene gun and electrophoresis method do ensure us the rapid and optimized DNA transfer but it doesn’t give us the guarantee of enhanced survival of the transformed cells. So, to minimize the cell damage or to increase the survival of tissues, the biological method is considered for gene transformation which contains the medium of high osmotic pressure which allows the useful DNA to be propelled inside the cell of. This technique was first utilized on yeast and to increase transformation rates in plants.

    2.2.1.    DNA Microinjection

This method involves the direct microinjection the selected gene construct which is extracted from the source organism into the target fertilized egg. This technique has been applicable to mammals as well. This results in the expression of an altered gene or a totally new gene in that specie. DNA insertion is a challenge in this technique because there are chances that the DNA will not attach to the host cell and thus there will be no expression. After the modification of egg, it is implanted back into the womb of a foster mother.

    2.2.2.    Embryonic stem cell-mediated gene transfer

Stem cells are the undifferentiated cells that have the potential to develop into any type of body cells either germline or somatic cells. So, in this technique, we first introduce our target gene into an in vitro culture of embryonic stem cells by using homologous recombination. Now we take an embryo at blastocyte stage of development and insert the somatic cells into it which leads to the formation of a chimeric organism. It is also used for gene inactivation commonly known as knock-out method.

    2.2.3.    Retrovirus-mediated gene transfer

In order to enhance the chances of gene expression, we tend to use a gene carrier called vectors which include plasmids and viruses. Retroviruses are the most common uses vectors as their ability to infect host cells is used as a tool. Resulting progeny is chimeric not all the offspring have virus integrated into their genome.

By: Rimsha Zafar

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