Genetic Engineering

Genetic engineering, recombinant DNA technology and biotechnology –  It is a set of techniques that are used to achieve one or more of three goals-

• to reveal the complex processes of how genes are inherited and expressed,
• to provide better understanding and effective treatment for various diseases and
• to generate economic benefits which include improved plants and animals for agriculture, and efficient production of valuable biopharmaceuticals.

The characteristics of genetic engineering possess both vast promise and potential threat to human kind. It is an understatement to say that genetic engineering will revolutionize the medicine and agriculture in the 21st future. As this technology unleashes its power to impact our daily life, it will also bring challenges to our ethical system and religious beliefs.

1.0Introduction

• Genetic engineering is the direct modification of an organism’s genome, which is the list of specific traits (genes) stored in the DNA. 
• Changing the genome enables engineers to give desirable properties to different organisms. 
• Organisms created by genetic engineering are called genetically modified organisms (GMOs)

2.0Genetic Engineering and Human Health 

The creation of the first engineered DNA molecule through splicing DNA fragments of two unrelated species together was made public in 1972. Soon followed were a whole array of recombinant DNA molecules, genetically modified bacteria, viruses, fungi, plants and animals. The birth of “Dolly”, the first mammal ever cloned from an adult body cell, has elevated the debate over the impact of biological research to a new level. Furthermore, a number of genetically modified organisms have been commercially released since 1996. Today, it is estimated that over 70% of US foods contain some ingredients from GMOs. 

Genetic engineering holds tremendous promise for medicine and human well-being. Medical applications of genetic engineering include diagnosis for genetic and other diseases, treatment for genetic disorders, regenerative medicine using pluripotent cells, production of safer and more effective vaccines and pharmaceuticals, the prospect of curing genetic disorders through gene therapy,  the list goes on. Owing to its potential to give humanity unprecedented power over life itself, the research and application of genetic engineering has generated much debate and controversy. Many human diseases, such as cystic fibrosis, Downs syndrome, fragile X syndrome, Huntington’s disease, muscular dystrophy, sickle-cell anemia, Tay-Sachs disease etc. are inherited. There are usually no conventional treatments for these disorders because they don’t respond to antibiotics or other conventional drugs. Another area is the commercial production of vaccines and pharmaceuticals through genetic engineering, which has emerged as a rapidly developing field. The potential of embryonic stem cells to become any cell/tissue/organ under adequate conditions holds enormous promise for regenerative medicine. 

3.0Transformation of dicotyledonous plants

Dicotyledonous plants are those which develop from two cotyledons in the seed. They can be recognized by the branching veins in their leaves. Dicots of commercial value include many horticultural plants such as petunias, and crops such as tobacco, tomatoes, cotton, soybean and potatoes. Petunias have been engineered to produce a range of attractive flower colors and patterns. Tobacco, due to its ease of transformation, initially became the workhorse of plant genetic engineering, but more recently the common wall or thale cress, Arabidopsis thaliana, has become very popular. It has the advantage of not requiring tissue culture during its transformation. Tomatoes have been transformed to delay their ripening, cotton to insect resistance and herbicide tolerance, soybeans to improved oil quality and herbicide tolerance, and potatoes to resist viruses.

4.0Transformation using Agrobacterium tumefaciens 

Agrobacterium tumefaciens is a plant pathogenic soil bacterium. It makes (tumefacient = swollen: Latin = tumefacere) a tumor on plants it infects and as these are often on the crown region where the stem meets the roots, the disease is called Crown Gall. Scientists were amazed to discover that the bacteria transfer part of their DNA to the plant nucleus where it becomes integrated into the plant genetic material. The transferred DNA, or T-DNA, is part of a large tumor-inducing (Ti) plasmid. The TDNA carries an onc (oncogenic) region, which, by coding for the production of plant growth hormones, results in the proliferation of plant cells forming a tumor or gall. It also codes for the production of unusual derivatives of arginine, such as nopaline or octopine, which the bacteria can use as growth substances. This bacterial-plant interaction is known as genetic colonization. It was not long after this discovery that scientists realized that the introduction of a foreign gene into the T-DNA would enable its transfer to the plant cell nucleus. This led to the development of plant transformation using a disarmed, onc-, version of the Ti plasmid that could transfer DNA into plants without causing the production of a tumor.  

The Ti plasmid is very large, in the order of 200 kb, and therefore unwieldy to work with in vitro. It was soon discovered that all that is required for a gene to be introduced into a plant are the 25-bp repeat sequences at the borders of the onc region, known as the left and right borders (LB and RB), and the virulence genes (vir) of the Ti plasmid. It was possible, therefore, to separate these in a system of binary vectors. Genetic manipulation is done in Escherichia coli on a small plasmid carrying a multiple cloning site (MCS) downstream of a plant promoter, and a gene coding for resistance to a herbicide or antibiotic that is toxic to the plant of interest, situated between the LB and RB. This plasmid is then transformed into a strain of A. tumefaciens carrying a disarmed Ti plasmid, which essentially consists only of the vir region and an origin of replication This strain of A. tumefaciens is then used to transform plants. The earliest species to be transformed was tobacco, Nicotiana tabacum, which rapidly became the model dicot plant. However, more recently the workhorse has changed to Arabidopsis thaliana which has a very small genome of 120 Megabases and is easier to transform. In order to transform tobacco, and most other dicots, leaf disks are cut and placed in a Petri dish containing a liquid medium. The A. tumefaciens strain is placed on the surface of the disks and co-cultivation carried out for 2-3 days. The cutting of the leaf disks results in the plant producing wound-response compounds, such as acetosyringone, which induces the virulence genes. The leaf disks are then transferred to selection media containing the herbicide or antibiotic of choice. This is often kanamycin as many binary vectors carry the neomycin phosphotransferase gene (NPTII) which codes for kanamycin resistance. Transformation occurs along the cut edges of the disks, resulting in the formation of callus tissue which carries the DNA between the LB and RB integrated at random into the plant genome. The callus tissue is then transferred to regeneration medium also containing kanamycin, which only allows transgenic plants, expressing kanamycin resistance, to develop. The whole process takes about three to four months. During the regeneration process care must be taken to inhibit the growth of Agrobacterium as false positive results could be due to the expression of the T-DNAcarrying genes in the bacteria rather than in the plant. This is often found despite the fact that the genes are expressed from eukaryotic promoters. Antibiotics such as carbenicillin or cefotaxime can be used to eliminate the bacteria but they are not always sufficient. Another strategy is to introduce into the T-DNA a GUS gene, coding for ßglucuronidase, which carries a plant intron. The enzyme is very easy to detect histochemically and fluorometrically and will only be correctly spliced if it is expressed within the plant and not in A. tumefaciens.