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RETROVIRUS MEDIATED GENE TRANSFER AND EXPRESSION CLONING
- 1. GURUNANAK INSTITUTE OF PHARMACEUTICALSCIENCE AND TECHNOLOGY A review on Retrovirus mediated gene transfer and expression cloning: A powerful tool in functional genomics Submitted by Name: Srishti Roy Stream: Bsc. (H) Microbiology Semester & Year: 6th and 2018-2021 University Registration No. : 183132410091 OF 2018-2019 University Roll No.: 31308418006 Under The Guidance Of: Dr Bhaskar Choudhury
- 2. SL.NO. CONTENT 1. Introduction 2.Structure of Retrovirus 3. Replication and Life Cycle of Retrovirus 5. Retroviral mediated pathogenesis 6. Retroviral interaction with human immune system 7. Retrovirus mediated gene transfer 8. Cloning vectors and different cloning vector systems 9. Expression vector v/s cloning vector 10. Retrovirus mediated expression cloning : applications 11. Retrovirus mediated expression cloning : advantages 12. Conclusion and Future Scopes 13. Reference 14. Acknowledgement
- 3. A retrovirus isa type of virus that inserts a copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. Once inside the host cell's cytoplasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern, thus retro (backwards). The new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as part of its own genome, transcribing and translating the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus. Although retroviruses have different subfamilies, they have three basic groups: The oncoretroviruses (oncogenic retroviruses), the lentiviruses (slow retroviruses) and the spumaviruses (foamy viruses). The Oncoretroviruses are able to cause cancer in some species, the Lentiviruses are able to cause severe immunodeficiency and death in humans and other animals, and the Spumaviruses are benign and not linked to any disease in humans or animals. The structure of retrovirus is as follows - a glycoproteinous membrane surrounding the whole body making it easily accessible to all the receptor binding proteins of the host cells,two strands of RNA genome enveloped within the cell (which even encloses a capsid) that have three enzymes: protease, reverse transcriptase, and integrase. In retroviruses, replication proceeds through an intermediate preliminary step, production of the substance termed as provirus. The proviruses can integrate at a great speed and randomly into the host cell chromosome and therefore becomes a cellular gene.
- 4. • The retroviralvirion is a spherical particle of about 80–100 nm in diameter. • It consists of two concentric outer circles of the lipid bilayer that contains the envelope protein complex embedded in it. The capsid proteins are hexagonal. • The retroviral envelope is composed of lipids and glycoproteins which serves three important functions: the lipid bilayer protects the retrovirus from the extracellular environment,helps the retrovirus to enter or exit the host cell through endosomal membrane trafficking and direct entry into the cells by fusing with the membranes. • In addition the virion contains two identical copies of a genomic RNA molecule, a tRNA primer for reverse transcription as well as small amounts of the products of the viral pol gene. While the simple retroviruses have only three genes, gag, pol and env .
- 7. • The humanimmune system needs to manage with various pathogens, ranging from RNA viruses to 30-foot-long tapeworms. The immune system retroviruses (ISRV) are defined as a retrovirus (HIV) whose target is T4-positive T-helper cells of the immune system which requires stimulation by antigens to procreate. • It was observed that generalized immune activation and increased amount of cytokines and immunoglobulins along with the progressive loss of CD4+ T-cells was reported during HIV-1 infection. Antiviral responses are characteristically marked by stimulation of type I interferon which restrain the viral replication by various mechanisms and activate the adaptive immune responses with the help of antigen presenting cells and also aids in developing memory and viral clearance.
- 8. The basic processof the retrovirus mediated gene transfer - Immediately following infection, the retrovirus produces a DNA copy of its RNA genome using reverse transcriptase. Completion of this process requires that the host cell undergoes the S phase of the cell cycle. Therefore, retroviruses effectively transduce only mitotically active cells. The DNA copy of the viral genome or provirus integrates randomly into the host cell genome, usually without deletions or rearrangements. Because integration is not by way of homologous recombination, this method is not used effectively for site-directed mutagenesis. https://youtu.be/WazDCNr3lB4 The process is explained here.
- 9. • It isthe central component of a gene cloning process. • A small piece of DNA into which a foreign DNA fragment can be inserted. The insertion of the fragment is carried out by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together. • https://youtu.be/ZYPnkKRa5Qs The vector is explained in this video
- 10. VECTORS BASIS APPLICATION PLASMIDNaturally occuring multicopy plasmids Subcloning and downstream manipulation, cDNA cloning and expression assays PHAGE Bacteriophage λ Genomic DNA , cloning cDNA cloning and expression libraries COSMID Plasmid containing a bacteriophage λ cos site Genomic library construction BAC( BACTERIAL ARTIFICIAL CHROMOSOME) E.coli F factor plasmid Analysis of large genomes YAC ( YEAST ARTIFICIAL CHROMOSOME) Saccharomyces cerevisiae centromere, telomere and autonomously replicating sequence Analysis of large genomes, YAC transgenic mice MAC ( MAMMALIAN ARTIFICIAL CHROMOSOME) Mammalian centromere, telomere and origin of replication Under development for use in animal biotechnology and human gene therapy
- 12. This process shownin the figure is used in stem cell therapy in various organisms and also to cure various gene related diseases. https://youtu.be/wuYNin4C jnY This video depicts how retroviruses are used in gene therapy
- 13. • Efficient retrovirus-mediatedgene transfer is useful to investigate functions of genes, particularly those with inhibitory functions in the control of cell growth because it is difficult to establish stable transformants that express such genes. • Alternatively, retrovirus vectors can be designed to express a dominant-negative form of a particular protein, the anti-sense cDNA, or the RNAi construct to investigate functions of a particular gene in a particular cell type. • Helps in the diagnosis of genetic disorders in organisms. • Improved retroviral systems are being developed both in packaging and vector components to facilitate applications to expression cloning and functional genomics.
- 14. In the past,insertional mutagenesis using retrovirus vectors was performed extensively; however, it was difficult to identify the responsive gene induced by the retrovirus because retrovirus integration could alter transcriptional activities of the genes within 20 to 30 kbp from the integration site. Recent genome information and expression profiling using gene chips and DNA microarrays have made it much easier to identify the responsive gene in insertional mutagenesis. Recently, even in vivo mammalian genetic screenings have been performed successfully using retrovirus-mediated insertional mutagenesis in the mouse. Most of the human genomes have now been sequenced, and about 30,000 genes have been identified. It is believed that retrovirus-mediated gene transfer and expression cloning will continue to be important tools to understanding the genome. In addition to expression cloning, retrovirus-mediated gene transfer also is useful for investigating gene functions and can be applied in high-throughput analysis of gene product in a variety of cells. Ongoing investigation on application of retroviruses in gene therapy and anti-cancer agents makes these type a widely studying group. The way retroviruses enter and target the specific cells and integrate itself into the host genome was very fascinating to the scientists globally, and these can be used as models to develop new vectors that could be employed in research. The reason is that this not only aids in developing antiviral, but also gives us idea where gained knowledge could be applied in other fields such as engineering and material sciences and to develop new technologies.
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- 17. This presentation isby far the most significant accomplishment in my life and it would not have been impossible without people who supported me and believed in my calibre. I would like to extend my gratitude and sincere thanks to my Mentor in this project Prof Bhaskar Chowdhury. He is not only a great teacher with deep vision but also most importantly a kind person. I would also like to thank our Director Sir , Prof Abhijit Sengupta , Principal-in-charge, Prof Lopamudra Dutta, Dr Swati Chakraborty, HOD Department of Life Science , and my other professors as well namely, Prof Tamalika Chakraborty, Prof Nirjhar Dasgupta, Prof Khaleda Firdous and Prof Basab Brata Dey. I sincerely thank each one of them for their exemplary guidance and encouragement. Their trust and support inspired me in the most important moments of making right decisions and I am glad to complete this project under his supervision. Last, but not the least, I want to thank my parents, who always supported me morally, and my fellow classmates for their constant support and help on completing my presentation.