27 - virus vectors II.docx

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Molecular Genetics and Microbiology
Alan Cochrane

MGY376 – March 30 th VIRUS VECTORS Modify/manipulate to transfer gene of interest Intracellular parasites that have evolved to efficiently transfer their host genes into cells Retroviruses – most predominant – can integrate their genes into the host genome – get stable gene expression – can also get high gene expression – can make pseudotype viruses – express and use correct promoter – Virus vector application - Retroviruses – long term stable expression – integrate their genes into host genome - Expression of short hairpin RNA – use retrovirus to express this which will knock out expression of specific gene - Gene therapy is continuing – introduce healthy gene in cells with defective gene, or for cancer treatment (introduce toxic gene to kill cancer cells); can tag a cell to see how things developed – - Vaccines – virus vectors as vaccines b/c can put in gene of interest and induce immune response in host SAFETY - High production of virus vector – target cell, 10^13 – high levels of virus - Delivery – majority of viruses do not target specific cells – want to hit only desired cells - How is expression controlled? - Tendency to activate or inactivate genes - Genes normally dormant – tendency to activate gene – lead to tumour formation Designing virus vector - When designing virus vector – - Any cell can be packaging cell, which packages proteins together to make virus particle - End up with replication complexes – viruses – - 1. Remove pathogenic viral genes – when in cell, get reduced pathogenicity – viral genes – some still packaged into viral particle, more likely to induce immune response – - 2. Remove packaging signal – - 3. Gene of interest is transgene, which has packaging signal – have gene of interest, promoters, packaging signal – only gene packaged in virus particle is transgene, left with virus vector with gene of interest Retrovirus vectors – - Have ability to pick up several genes - Some retroviruses able to infect - Can carry non viral genes - Helper retroviruses – taken transgene and to provide all the viral genes, used helper virus, which is replication competent virus – presence of helper virus, get virus vector with gene of interest but also replication competent virus - ADA enzyme required to convert the deoxyadenosine to deoxyinosine – when have this deficiency, converts deoxyadenosine and convert it to deoxyinosine – if mutation in enzyme, get build-up of two deoxyadenosine, which is shown to inhibit enzyme of ribonucelotide reductase – required for formation of two - The main cells that affect this are B cells and T cells – disrupted immune system- more prone to infection – B and T cells more sensitive to changes, undergo many cycles of division – even if weak mutation that inhibits ribonucleotide will have dramatic effect on growth - ADA treatment – take T, B cells out of patient – introduce healthy copy of gene using retrovirus using target cell and put the cell back into the patients - If treatment successful, leads to supplementing with recombinant enzyme – Designing retrovirus vector - Two stages of retrovirus lifecycle – one, entry – when genes are integrated - Then replication stage, whre gene is transcribed – get translation nand viral proteins assembled - Entry stage is virus vector with gene of interest – when develop a vector – need all the proteins required to go through the stage – need protein to make transgene – retrovirus has envelope protein, core protein, has genome, and that envelope protein initial stages of bind
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