Essay: Gene Transferring Techniques in Molecular Biology: Direct and Indirect Methods

Molecular Biology is a fascinating subject with full of surprises. I’m saying that because even it is still a recently evolved subject, it has achieved some extraordinary milestones in a very brief time. Gene transferring technique is also such a topic that brings the surprise element to the table. First let’s see what is meant by Gene Transferring Technique. It is the addition of novel genes or DNA segments into a recipient organism by a doner organism. Later these DNA becomes a permanent segment of the recipient genome and its viable seeds would inherit the respective characteristics to the offspring.
Early scientists used conventional breeding methods to achieve this goal to some extent. But there are few issues with the conventional gene transfer methods. First, they are only applicable for sexually compatible gene pool. And it is a time-consuming process. We may have to wait for completion of natural selection processes for several generations of the recipient organism. As the development of the technology, Genetic Engineering methods also immerged and evolved. So now we have the technology to transfer genes between sexually compatible organisms (cisgenesis) as well as sexually incompatible organisms (transgenesis) in a lesser amount of time.
When doing genetic modifications in plants There are two components which are essential for the stable expression of foreign genes. Those are the Expression Cassette and the physical, chemical, or biological method that using to deliver the novel gene into the recipient genome. Expression Cassette is a collection of specific sequences which would be carried into the recipient nucleus. It must contain the gene of interest and its corresponding promoter and terminator sequences, plant selectable marker and its corresponding promoter and terminator sequences, and some non-coding sequences in-between. Construct must be designed with unique restriction enzyme sites flanking the transgenic cassette. Then it would be easier to cutout the cassette from the vector using a single restriction enzyme.
Now let’s see what are the methods that we can use to transfer genes from one organism to another. Mainly methods could be either direct or indirect. Direct methods do not use biological vectors to transfer genes. Such methods use physical or chemical processes to insert genes into recipient cells. Using Polyethylene Glycol (PEG) is such a chemical method of gene transferring. Protoplasts which treated with PEG can rapidly abstract and integrate DNA into their genome. Electroporation is a physical method which applies a high voltage shock to the medium which contain DNA sequences and recipient cells. This can result small pores in the cell wall and DNA can easily migrate into the cells using those pores. Particle bombardment is also a physical method which use DNA coated gold or tungsten particles for transformation. When these particles shoot towards the plant tissue using a gene gun, they go through the cell walls, cell membranes and finally integrated to the recipient genome.
As direct methods scientists use Agrobacterium mediated transformations for genetic modifications. Here they can use co-integrated vectors or binary vector systems to achieve the goal. Basic steps of plant transformation of Agrobacterium as follows. First the gene of interest is isolated and develop the vector or vector system. Then the vector containing Agrobacterium is co-cultured with the suitable explant. After the completion of the transformation explants grow under suitable antibiotics to prevent the growth of Agrobacterium. Finally, should select transformed plant cells to regenerate plants.
The applications of the gene transfer technology in plants are mainly focusing on crop development. It has become such an important method to develop plants which are tolerant to extreme environmental conditions, plants with high nutritional value, plants with a high yield, plants which are resistance for herbicides, pests, fungal and virus diseases etc. On the other hand, gene transferring is important to study molecular aspects of basic plant processes. Now let’s see some examples for such applications in plants in details.
First let’s talk about a method that can develop protection against tobacco mosaic virus in transgenic plants. Transgenic plants that express the tobacco mosaic virus coat protein (TMV-CP) gene. The protection is enhanced by the presence of antisense RNA to the viral RNA. In eukaryotes no naturally occurring antisense RNA which participate in gene regulation is not reported thus far. But it can be occurred in prokaryotes. The function of antisense RNA is hybridizing with the viral RNA and preventing the process of translation. Scientists use two methods to introduce antisense RNA into recipient cells. When use the micro-injection method and inject antisense RNA directly to the cytoplasm, has more protection an efficiency than transferring using a vector. Where the antisense RNA expressed in the host genome is mostly limited to the nucleus and transported inefficiently to the cytoplasm.
To obtain the protection, various lengths of cDNA that encodes the antisense RNA, which are complementary to 3’ region of tobacco mosaic genome were inserted into a pMON316 plasmid under the Cauliflower mosaic virus 35s promoter. These plasmids then introduced to the Agrobacterium tumefaciens bacteria and co-cultivated with the leaf-disks of tobacco plants. Then the transgenic plants were regenerated. Experiments shows that such plants were more tolerant for tobacco mosaic virus at low concentrations than the plants not having transgenes.
To make tobacco plants tolerant to herbicide N-phosphonomethylglycine (glyphosate) scientists introduced mutant aroA gene of Salmonella typhimurium to the plant genome. Glyphosate is a broad-spectrum herbicide which could be infected both weed and crop plants. By this gene transfer the sensitivity of crop plants to the herbicide is decreased. The cellular function of the glyphosate is to inhibit the action of 3-phosphosikimate 1-carboxyvinyltransferase which catalysis the synthesis of 5-enol-pyruvylshikimate 3 phosphate from phosphoenolpyruvate and shikimate 3-phosphate. This inhibition results starvation of aromatic amino acids, accumulation of shikimate, and finally the cellular death.
Tolerance for glyphosate can be achieved by either over synthesizing 3-phosphosikimate 1-carboxyvinyltransferase or control the inhibition process glyphosate-resistant EPSP synthase enzyme. In this method aroA gene of Salmonella typhimurium is chosen. To construct the vector system Agrobacterium tumefaciens Ti-plasmids ware used. Isolated aroA genes of Salmonella typhimurium are sequenced. First construct is consisting of an aroA gene fused to octopine synthetase gene promoter. Second construct is consisting of mas-aroA, a hybrid gene fused under mannopine synthetase gene. These two sequences cloned into pPMG55 nas pPMG85 plasmids respectively. After both of these plasmid vectors co-integrating with pRiA4, strains harbored by them are co-cultivate with tobacco leaf disks. To examine whether mutant aroA gene products enhance the tolerance of tobacco plants to glyphosate, the offspring of the transgenic plants can be expressed to the herbicide and comparing the percentage of killed plants with a control. Such experimental results have showed that plants have developed a good tolerance and it depends on the expression level of the transgene.
When talking about the applications of transgenic plants, now scientists are more focused on using them in human health care(pharmaceutical) sector. Reported first clinical trial of such plant based antibodies was the CaroRXTM . It contained SIgA antibodies produced in transgenic tobacco plants. It had the ability to prevent the action of Streptococcus mutansn and reduce oral infections. Also, scientists have produced transgenic soybeans that synthesize anibodies against HSV-2 virus. These antibodies have shown successful results in mice by developing an immune protection against Herpes.
A research group of Stanford University has produced a tumor-specific vaccine using plant virus based transient expression system. Modified tobacco mosaic virus vectors contain segments which will be encoded into 38C13 scFv protein were transferred into Nicotina benthamiana plants and the transgenic plants synthesize high number of proteins into the apoplast. Isolated proteins then vaccinated to mice suffering from 38C13 tumor. The study showed that the rapidly produced protein can develop a protection and can significantly reduce the death rate.
Edible vaccine production is also a topic that highly focused in genetic engineering. After integrating the transgene into the plant genome by using a direct or an indirect method it will produce antigens and will protect them using cell walls. Few examples for such incidents are potato plant used to producing vaccines against tetanus, diphtheria, hepatitis B and Norwalk virus. Using potato is easy to transformation and propagation. No need of refrigerators. But main drawback is the denaturation of antigens while cooking. Edible vaccines based on fruits like banana and tomato can solve that problem. Those fruits can eat as raw food and protected by the cell walls by digestive enzymes. An effective vaccine against SARS caused by coronavirus was earlier produced by genetic modifications in tomato plant genome. Other than that tomato plant is used to produce edible vaccines against Vibrio cholera B toxin, Alzheimer’s disease, pneumonia, septicaemia etc. These are only few examples for plants that used to produce edible vaccines. Other than that, plants such as Rice, Lettuce, Tobacco, Carrots are also used in industry for produce edible vaccines.
Nutritionally enhanced food crops are also an important application in transgenesis which address the issue of malnutrition worldwide. Normally, these vitamins and minerals adding to crops through biofortification practices. But this method cannot fulfil the goals set for food production. The other method is using the genetic engineering methods. This is the easiest method among these two because even it involved a certain amount of effort and time and the beginning it could be maintained at a lower cost and labor.
Now let’s look at few examples where plants are genetically modified to enhance the nutrition value. Most used plant for biofortification is rice due its high consuming all around the world. Vitamin A deficiency is an issue that cause eye damage in millions of children, approximately half a million becomes blind annually. So, the rice plants were modified to produce β-carotene, the precursor molecule of Vitamin A biosynthesis. Genes of daffodil and Erwinia uredovoia bacterium are used in this process. The traits are transferred into high yielding local cultivars such as GR2 and execution of genes may result golden colored grains due to high amount of β-carotene. So, these rice plants are named as golden rice. Studies shows that β-carotene produced by golden rice plants are just as effective as natural β-carotene.
Other than that rice is also been engineered to prevent iron and folate deficiency by adding phytase and improving iron storage, synthesizing essential amino acids by RNAi silencing, reducing phytic acid levels, which binds to zinc like minerals that cause nutritional consequences by transferring Aspergillus niger gene which synthesizing phytase enzyme etc.
Crop yield increasing and making plants drought stress tolerant in stressful conditions is another use of genetic modifications in plants. In certain areas of the world has drought conditions almost throughout the year. Crops in such areas should have resistance for extreme conditions. But a newly introduced plant would take generations for be naturally selected and develop such traits. But using genetic modifications now it can achieve the same goal within a limited amount of time. These plants most often contain one or more transcription factors, which response to drought stress by controlling the gene expression. Now let’s see some examples for transcription factors.
DREB (Dehydration Responsive Element Binding) helps to enhance drought tolerance in crop like wheat. TaDREB1 transcription factor is activated in drought conditions in such plants. It also resistance for water stress, WRKY2 factor is a repressor that controls the plant growth in both abiotic and biotic stress. HDG11 factor regulate the expression of ABA synthesis pathway. Transgenic plants that overexpressing corresponding gene studied to be exhibit lower water loss and higher yield in stressful conditions. TaSH1, NAC and bZIP2 are some of other factors that can tolerate the drought conditions by transferring into plant genome.