Vectors for gene transfer in animals: Retro virus

Retro virus mediated gene transfer in animals.

1. Vectors for gene transfer in animals: retrovirus Presented by :- Khushbu Kumari Roll no. 2 01631 M.sc Biotechnology

2. Basic terminology Viral vector – A synthetic construct containing given viral sequences determined to transfer genetic information into the target cell. Transfection – Transfer of genetic information by a non-viral system. Transduction – Transfer of genetic information by a viral vector (genetic information is packaged in the viral particle). Transient – DNA is not integrated into the genome of host cell, genetic modification is temporary. Provirus – Viral genome integrated into chromosome of the host cell. Gene transfer -The introduction of new DNA into an existing organism's cell, usually by vectors such as plasmids and modified viruses. Cells may be modified ex vivo for subsequent administration to humans, or may be altered in vivo by gene therapy given directly to the subject.

3. Vectors used for gene transfer in animals Adenoviruses These are medium-sized (90–100 nm), nonenveloped (naked) icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. Adeno- associated virus These viruses are not genetically related to adenovirus but it is so called because it was first discovered as a contaminant in an adenovirus isolation.AAV are small viruses that infect humans and some other primate species.They are small (20 nm) replication-defective, non enveloped viruses and have linear single- stranded DNA. Baculovirus Have rod shaped capsid and large dsDNA genomes.Mostly infect insects but can also infect some mammalian cells.

4. Retrovirus Retroviruses are animal viruses with two identical strands of RNA in their virion. Retroviruses used as vectors to transfer genetic material into the host cell. When these viruses infect a host cell, the viral RNA is reverse transcribed in the cytoplasm to make linear double-stranded DNA, which is transported into the host cell nucleus and integrates into a chromosome directly without any change in its original linear form. The viral genes are expressed from this integrated form of DNA, the provirus, and the progeny viruses are produced from the infected host cell as a result of proviral gene expression.

5. Life cycle of Retrovirus The genome of the retrovirus consists of two identical copies of RNA. After infection, the reverse-transcribed DNA of integrates into the host cell chromosome. The viral genes are expressed from this integrated form of DNA,provirus. Following transcription, both full-length and spliced RNAs are exported out of the nucleus, and then translation follows. Because spliced RNAs are devoid of encapsidation signal sequence, only non spliced full-length RNAs are packaged by viral proteins, and the resulting virions are budding out of cells.

6. Retrovirus Vector Systems Simple retroviruses have three trans-acting protein-coding genes: gag, pol, and env. Group-specific antigen (gag) codes for core and structural proteins of the virus, polymerase (pol) codes for reverse transcriptase, protease and integrase, and envelope (env) codes for the retroviral coat proteins. To construct a virus that can be used as a trans gene vector, only cis-acting DNA sequences should be retained with the gene sequence of interest. Consequently, the resultant virus is replication defective, but with the coordination of trans-acting functions provided from another source (i.e., a helper cell or packaging cell), the virus becomes infective to target cells.

7. Steps for gene transfer in animals Step 1 Step 2 Target gene

8. Step 3 Step 4

9. Step 5

10. Step 6 Step 7

11. Result

12. Advantages and Disadvantages of Retrovirus Advantages E ase of manipulation for insertion of the therapeutic gene; A bility to stably integrate into the target cell genome; R elatively high titer of the recombinant retroviruses; A wide range of target species and cells that can be infected without any apparent adverse pathology; and R elatively simple procedure for preparation of the recombinant virus. Disadvantages R equirement for cell division for integration, limiting their in vivo applications; and R andom integration into host chromosome, resulting in possible insertional mutagenesis or oncogene activation.

13. References https://www.intechopen.com/chapters/49607 https://www.slideshare.net/rahulgoswami454/gene-transfer-in-animals https://www.sciencedirect.com/science/article/pii/B978012410490700006 https://youtu.be/Uemz6YkWQN4 https://link.springer.com/chapter/10.1007/978-0-387-70909-3_5