Genetic Engineering

Genetic Engineering:

For ages human beings have  been using sexual  and asexual  reproduction techniques in plant and animal  breeding  for creating hybrids and for improving  the existing  stock of plants and animals. These techniques  try to improve the genetic  stock available  to the mankind. These traditional  techniques  have number of limitations llike propagation of  undesirable techniques along with desirable techniques. 

  Genetic Engineering, on the other hand, tries to achieve the same purpose by tinkering  with the DNA of the living organisms. These techniques like recombinant DNA technology, gene transfer, gene cloning, etc.,also help  in dealing  with the  shortcoming of the traditional breeding  techniques. 

 Recombinant DNA Technology:

The recombinant DNA technology  is the technology used for creating new DNA  from the existing DNA. 

 The genetic  engineering  required  the following  three basic steps:

1): Identification  of DNA with desirable genes


2): Introduction of the identified DNA into the hos

3): Maintenance  of introduced DNA in the host and transfer of the DNA  to its progeny 

In addition,  the genetic engineering  requires the following  tools:

Restriction enzymes  : Restriction enzymes  are the enzymes  belonging  to the class of enzymes known as nucleases. These enzymes  are further divided  into two types:

1): Exonucleases: These are the enzymes  that remove nucleotides  from the end if the DNA. 

2): Endonuclease: These are the enzymes  that cut at specific position within the DNA.
These enzymes  inspect the length  of  the DNA sequence,  identify  the specific  recognition sequence, bind to it and cut each of the two strands of the DN at specific points.


Gel electophoresis: Both genetic engineering  and creation of recombinant DNA require DNA fragments . The restriction enzymes  are used for cutting  the DNA and for obtaining fragments of DNA fragments  created by the  use of restriction  enzymes  have negative charge. This charged is used for forcing  them to separate. For this, a medium or matrix , such as, agarose  is used.

Cloning  Vectors: The DNA fragments  obtained  by gel electrophoresis  cannot be introduced into living hosts as they are. They will not multiply in the host because  they will be alien  to  the host. This is where cloning  vector comes in. Cloning  vector is a DNA molecule ( normally a plasmid or bacteriophages) that is capable of self/ replication  in the host. The DNA fragments  are attached to the cloning  vector and this is then  introduced into the host.


  

GENOME EDITING 

Genome editing, also known as genome engineering or gene editing, is a sort of genetic engineering that involves inserting, deleting, modifying, or replacing DNA in a living organism’s genome.

Genome Editing  Techniques:

The core technologies now most used to facilitate genome editing, are

●Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9): CRISPR is the DNA-targeting component of the system, and it is made up of an RNA molecule, or ‘guide’, that is engineered to attach to certain DNA bases via complementary base-pairing.



●CRISPR-associated protein 9 (Cas9) is the nuclease component that cuts the DNA.



●The CRISPR-Cas9 genetic scissors were discovered by Emmanuelle Charpentier and Jennifer A. Doudna, who won the Nobel Prize in Chemistry in 2020.



Transcription activator-like effector nucleases (TALENs): 


Transcription activator-like effector (TALE) domains make up the DNA-binding domain of TALENs. The nuclease portion of TALENs, like ZFNs, is usually a FokI nuclease. 

Zinc-finger nucleases (ZFNs): 

ZFNs are fusions between a custom-designed Cys2-His2 zinc-finger protein and the cleavage domain of the FokI restriction endonuclease. FokI cleavage domain, which cuts DNA within a five- to seven-bp spacer sequence that separates two flanking zinc-finger binding sites. 

Homing endonucleases or mega nucleases:

 Homing endonucleases, also known as mega nucleases. These enzymes make extensive sequence-specific contacts with their DNA substrate. However, unlike ZFNs and TALENs, the binding and cleavage domains in homing endonucleases are not modular. This overlap in form and function makes their repurposing challenging, and limits their utility for more routine applications of genome editing.

T-Cell (CAR-T) Therapy


Chimeric Antigen Receptor (CAR) T-cell therapy involves genetic modification of a patient’s autologous T-cells to express a CAR specific for a tumor antigen.

It is followed by ex vivo cell expansion and re-infusion back to the patient.   

CARs are fusion proteins of a selected singlechain fragment variable from a specific monoclonal antibody and one or more T-cell receptor intracellular signaling domains.


This T-cell genetic modification may occur either via viral-based gene transfer methods or non-viral methods, such as DNA-based transposons, CRISPR/ Cas9 technology or direct transfer of in vitro transcribed-mRNA by electroporation. 



Significance of Genome Editing 

These techniques affect different areas such as disease management, basic biomedical research, agriculture, and environmental sciences. They could also be used to customize human characteristics for extra-therapeutic enhancement purposes. 

Concerns associated with Genome Editing

Safety: Due to the possibility of off-target effects (edits in the wrong place) and mosaicism (when some cells carry the edit but others do not), safety is of primary concern.

The International Summit on Human Gene Editing, generally agrees that until germline genome editing is deemed safe through research, it should not be used for clinical reproductive purposes; the risk cannot be justified by the potential benefit.

Informed Consent it is impossible to obtain informed consent for germline therapy because the patients affected by the edits are the embryo and future generations.

Many people have moral and religious objections to the use of human embryos for research.


● Potential loss to diversity : Diversity in all species of animals is a key to evolution on earth. Genetically engineering our species will have a detrimental effect on genetic diversity.


● No Guarantee  of Food Security: 
In India, there is a moratorium on Bt brinjal because there is no scientific consensus on its safety and efficacy.

Three parent Baby 

Techniques to create ‘three-parent babies’ seek to offer mothers a way to have a child without passing on metabolic diseases caused by faulty mitochondria.

Researchers do this by exchanging the diseased mitochondria of a prospective mother with those of a healthy, unrelated donor: the ‘third parent.’


In addition to DNA in the nucleus, some DNA is also present in the mitochondria.

●This technology uses the technique of Pronuclear Transfer.

In pronuclear transfer, a zygote is created by first fertilizing the mother’s egg with the father’s sperm.

☆The donor egg is then fertilized and has had its own nucleus removed before the pronuclei of the egg and sperm are removed from the zygote and put into the donor egg (a pronucleus is the nucleus of the egg or sperm at the stage of fertilization prior to nuclear fusion).

☆The donor egg’s zygote is subsequently inserted into the mother’s uterus.

☆During fertilization the nuclear DNA is formed with 46 chromosomes (i.e., 23 from mother & 23 chromosomes from the father).

☆The Mitochondrial DNA has only one chromosome and it codes for only specific proteins responsible for metabolism

☆Mitochondrial DNA is inherited only from the mother & thus it is more effective to trace human ancestry.

1).In the context of recent advances in human reproductive technology, ‘Pronuclear Transfer” is used for (2020)

(a)Fertilization of egg in vitro by the donor sperm

(b)Genetic modification of sperm producing cells.

(c)Development of stem cells into functional embryos

(d)Prevention of mitochondrial diseases in offspring.



Somatic cell Nuclear Transfer:



1): Somatic Cell nuclear Transfer  (SCNT) technique  in which the nucleus of the somatic ( body) cell is transferred to the cytoplasm of an enumerated egg ( an egg that has had it's own nucleus removed ).Once inside the egg, the somatic nucleus is  reprogrammed by egg cytoplasmic factors to become a zygote ( fertilized eggz) nucleus.




2)The egg is allowed to develop to the blastocyst stage, at which point a culture of embryonic stem cells (ESCs) can be created from the inner cell mass of the blastocyst.

For example, Cloning of Dolly Sheep

1).Dolly was cloned from a cell taken from the mammary gland of a six-year-old Finn Dorset sheep and an egg cell taken from a Scottish Blackface sheep.

2).She was born to her Scottish Blackface surrogate mother on 5th July 1996.

3)Dolly’s white face was one of the first signs that she was a clone because if she was genetically related to her surrogate mother, she would have had a black face.

4Dolly’s DNA came from a mammary gland cell, and she was named after the country singer Dolly Parton.


1.What is the application of Somatic Cell Nuclear Transfer Technology? (2017)
    (a)
    Production of bio larvicides
      (b)
      Manufacture of biodegradable plastics
        (c)
        Reproductive cloning of animals
          (d)
          Production of organisms free of diseases

          Genetically Engineered Insulin

          Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs.


          1).Insulin from an animal source, though, caused some patients to develop allergy or other types of reactions to the foreign protein.


          2).In 1983, Eli Lilly, an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E. coli to produce insulin chains.


          3)Chains A and B were produced separately, extracted, and combined by creating disulphide bonds to form human insulin.

          Gene Therapy

          Gene therapy is a technique for treating genetic problems that includes replacing faulty genes with healthy ones.


          It is a way of introducing DNA into human cells that is done artificially.

          Gene therapy can be divided into two categories.

          Somatic Gene Therapy (SGT)


          This type is most seen in the somatic cells of the human body.


          This is specific to a particular person, and the damaged cells will only be replaced with healthy cells in that person.


          Therapeutic genes are introduced into the somatic cells of the human body using this procedure.

          This approach of gene therapy is thought to be the best and safest.

          Gene Therapy in the Germline


          It happens in the human body’s germline cells.


          Generally, this approach is used to address disease-causing genetic abnormalities that are handed on from parents to their children.


          The procedure entails inserting healthy DNA into the cells that produce reproductive cells, eggs, or sperms.

          Application in Bioenergy

          Biofuels derived from biomass are renewable and sustainable energies with the potential to replace fossil fuels.

          Biotechnology can help to speed up the selection of varieties that are more suited to biofuel production – with increased:


          1) biomass per hectare,

          2) increased content of oils (biodiesel crops) or

          3)fermentable sugars (ethanol crops), or

          4)improved processing characteristics that facilitate their conversion to biofuels.

          Utilization of microbial fuel cells is found to be useful for sustainable bioenergy synthesis via completing the wastewater treatment processes with electric energy synthesis.

          Environmental Biotechnology

          Environmental biotechnology, specifically, refers to the use of procedures to safeguard and restore the environment’s quality.

          Mycorrhizal biotechnology: Mycorrhizae are indigenous to soil and plant rhizosphere and potential tools for sustainable agriculture. They enhance the growth of a root system and even of an entire plant and often control certain plant pathogens.


          1). Numerous studies have documented the fundamental importance of mycorrhizal symbiosis and other microbial systems in reclamation and restoration of contaminated and disturbed ecosystems

          Bioremediation:

          Bioremediation is the process of using microorganisms to remove or detoxify toxins from soils, water, or sediments that would otherwise be harmful to human health.


          ■ Bioremediation is also known by the terms biotreatment, bioreclamation, and biorestoration.

          ■ Microorganisms are employed in sewage treatment plants to remove typical pollutants from wastewater before it is discharged into rivers or the sea.

          ■ Lindane (Hexa-Chlorocyclohexane) bioremediation technology has been developed

          Phytoremediation:

          Phytoremediation is a bioremediation process that uses various types of plants to remove, transfer, stabilize, and/ or destroy contaminants in the soil and groundwater.


          ■Phytoremediation treatment processes have been developed for the degradation of dyes from textile industrial effluent.

           ■The study showed that the developed process has the potential for textile dyes and effluentreatment. 
           

          phyto _ degradation: In this process, plants metabolize and destroy contaminants within plant tissues.

          Phyto _ vllatolization: In this process, plants take up water containing organic contaminants and release the contaminants into the air through their leave.

          Biosensors


           A biosensor is an analytical device that converts a biological response into a physical, chemical, or electrical signal. The biosensors can be designed to be very selective, or sensitive to a broad range of compounds. For example, a wide range of herbicides can be detected in river water using algal-based biosensors; the stresses inflicted on the organisms being measured as changes in the optical properties of the plant’s chlorophyll. 

          Gene Silencing 


          Gene silencing is the regulation of gene expression in a cell to prevent the expression of a certain gene. When genes are silenced, their expression is reduced. Ex: the researchers designed two small RNA molecules that silence the fungal genes which produce aflatoxin in Groundnut. When genes are knocked out, they are completely erased from the organism’s genome and thus, have no expression. 

          Applications: 


          Specific gene silencing using RNAi in cell culture. Cancer treatments RNA interference has been used for applications in biotechnology. Useful in epigenomic analysis and clinical application of molecular diagnosis. Neuro-degenerative disorders treatment. 

          COLOR CLASSIFICATION OF BRANCHES OF BIOTECHNOLOGY: 


          Gold biotechnology or Bioinformatics:

           Computational Biology à address biological problems using computational techniques. 

          Red Biotechnology: Biopharma à relates to medicine and veterinary products.

           White Biotechnology: Industrial Biotech à to design more energy efficient, low resource consuming products. 

          Yellow Biotechnology: Biotech in the Food Industry. 

          Grey Biotechnology: Environmental applications to maintain Biodiversity. 

          Green Biotechnology: Emphasizes on Agriculture interests. 

          Blue Biotechnology: based on use of marine resources. 

          Violet Biotechnology: deals with law, ethical and philosophical issues of biotechnology. Dark Biotechnology: associated with bioterrorism and biological weapons.


          Synthetic Biology


           Synthetic biology is a field of science that involves redesigning organisms for useful purposes by engineering them to have new abilities. Redesigning organisms so that they produce a substance, such as a medicine or fuel, or gain a new ability, such as sensing something in the environment, are common goals of synthetic biology projects. Some examples of what scientists are producing with synthetic biology are: Microorganisms harnessed for bioremediation to clean pollutants from our water, soil, and air. Rice modified to produce beta-carotene, a nutrient usually associated with carrots, that prevents vitamin A deficiency. Yeast engineered to produce rose oil as an ecofriendly and sustainable substitute for real roses that perfumers use to make luxury scents. 

          Difference between Synthetic Biology and Genome Editing


           In synthetic biology, scientists typically stitch together long stretches of DNA and insert them into an organism’s genome. These synthesized pieces of DNA could be genes that are found in other organisms or they could be entirely novel. In genome editing, scientists typically use tools to make smaller changes to the organism’s own DNA. Genome editing tools can also be used to delete or add small stretches of DNA in the genome.


          Bioinformatics


           Bioinformatics can be defined as “the application of computational tools to organize, analyze, understand, visualize and store information associated with biological macromolecules.” Bioinformatics has also been referred to as ‘computational biology’. However, strictly speaking, computational biology deals mainly with modeling of biological systems. 

          The main components of bioinformatics are: 


          1. the development of software tools and algorithms and 

          2. the analysis and interpretation of biological data by using a variety of software tools and particular algorithms.


          Bioinformatics is essential for management of data in modern biology and medicine.

           PRELIMS QUESTION 

          1. In the context of the developments in Bioinformatics, the term ‘transcriptome,’ sometimes seen in the news, refers to –
          (2016) 

          (a) a range of enzymes used in genome editing 

          (b) the full range of mRNA molecules expressed by an organism

           (c) the description of the mechanism of gene expression 

          (d) a mechanism of genetic mutations taking place in cells 

          Genomics and Proteomics


           The term “genomics” describes the study of an organism’s complete set of genes. The entirety of an organism’s genetic code, primarily composed of DNA, is included in its genome. In genomics, high throughput methods are used to map, sequence, and analyse genomes.

           Functional and structural genomics are the two primary subfields of genomics.

           While the function or role of the genes in controlling metabolic activities is examined in functional genomics, the structure and relative placements of the genes are studied in structural genomics. 

          Proteomics is the study of all the proteins that a cell produces. 

          The entire collection of proteins that a cell produces is known as the proteome. High throughput techniques are used in proteomics to characterise the 3D structure and the function of proteins.

          Significance of Genomics and Proteomics


          The results of genomics and proteomics increasingly promise the potential for future widespread adoption in medicine and biology. Simultaneous measurement of many mRNA levels now can reveal patterns of gene expression for an organism or a tissue under various conditions that can then be compared, pointing to genes characteristic of certain states or reactions. For example, distinct sub-types of large-cell lymphomas, with quite different responses to chemotherapy, can be distinguished from one another by measuring mRNA expression patterns, thereby providing a means of directing therapy. Likewise, serum samples can be decomposed into a spectrum of proteins, looking for patterns—referred to as biomarkers—of a particular disease, opening up the possibility for early detection and diagnosis. Genomic Surveillance: Genomic surveillance is the technique of continuously tracking diseases and comparing and contrasting their genetic make-up to spot potentially dangerous mutations. Stronger pandemic and epidemic preparedness and response depend on genomic surveillance. 

          Xenotransplantation 


          xenotransplantation is “any procedure that involves the transplantation, implantation or infusion into a human recipient of either 

          (a) live cells, tissues, or organs from a nonhuman animal source, or

           (b) human body fluids, cells, tissues or organs that have had ex vivo contact with live nonhuman animal cells, tissues or organs.”

          Xenotransplantation is seen as an alternative to the clinical transplantation of human organs whose demand around the world exceeds supply by a long distance. 

          Xenotransplantation, if found compatible overall, could help provide an alternative supply of organs to those with life-threatening diseases. 

          Pig heart valves have been used for replacing damaged valves in humans for over 50 years now. 

          The molecular incompatibility between pigs and humans can trigger several immune complications after the transplant, which might lead to rejection of the xenograft.

           To prevent that situation, genetic engineering is used to tweak the genome of the pig to ‘disguise’ it, so that the immune system of the human recipient fails to
          recognise it, and the reactions that lead to xenograft rejection are not triggered.

           GOVERNMENT INITIATIVES FOR BIOTECHNOLOGY 


          National Biotechnology Development Strategy 

          The Department of Biotechnology (DBT), Government of India, announced the First National Biotechnology Development Strategy in September 2007. 

          In 2015, DBT announced “The National Biotechnology Development Strategy-2015-2020” and later in 2020 for National Biotechnology Development Strategy 2021-2025. 

          Key elements of Strategy-II are as follows:


           Empower, scientifically and technologically, India’s incomparable human resource; 

          Create a strong infrastructure for research, development, and commercialization for a robust bioeconomy; 

          Establish India as a world class bio-manufacturing hub for developing and developed markets. 

          BIRAC 


          Biotechnology Industry Research Assistance Council (BIRAC) is a not-for-profit Section 8, Schedule B, Public Sector Enterprise, set up by the Department of Biotechnology (DBT). It is an Interface Agency to strengthen and empower the emerging Biotech enterprise to undertake strategic research and innovation, addressing nationally relevant product development needs.






























































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