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Reporter gene and gene fusions T.Y.Bsc
- 1. The Mandavi Education Society T.Y.Bsc(Microbiology) Krishna prajapati Teaching Assistance(Microbiology)
- 2. Reporter gene and gene fusions
- 3. Introduction DNA manipulation has revolutionary the study of gene regulation. A coading sequence from one source(the reporter)may be fused to a regulatory region from another source. Such gene fusions are often used in studying gene regulation especially where assaying the level of the natural gene product is difficult or time consuming. They may also be used to increase expression of desired gene product.
- 4. Reporter genes The key property of reporter gene is that it encodes a protein that is easy to detect and assay. Reporter gene are used for variety of purposes. They may used to report on the presence or absence of particular genetic elements(such as plasmid)or DNA inserted within the vector. They can also fused to other genes or to promoter of other genes so that gene expression can be studied.
- 5. LacZ gene The first gene to be used widely as reporter gene was lacZ, which encodes the enzyme 硫-galactosidase. Cells expressing 硫- galactosidasecan be detected by easily by their color on indicater plates that contain the artificial substrates Xgal ,which cleaved by 硫-galactosidase to yield blue colour.
- 6. Blue white screening .
- 7. Conti..
- 8. Green flourescent protein(GFP) Another widely used reporter gene encodes luciferase. This enzymes makes cells expressing it luminescent. colonies containing the reporter system can be detected by on agar plate by their luminscense against a large background of other colonies.
- 9. Conti.. However,the expression of luciferase depends on more than one gene beacause several accesary factors are needed. By contrast, the green fluroscent protein needs no accessary factors and is widely used as reporter Gene for was GFP was originally cloned from the jelly Aequorea victoria,the GFP protein may be expressed in most bacterial cell. It is stable and causes little or no disruption of host cell metabolism.
- 10. Conti If expression of cloned gene linked to that of GFP, expression of GFP signals that the cloned gene has also been expressed. Recent advance in fluorescent labeling now allow the simultaneous use of multiple fluorescent markers.
- 11. Some like that..
- 12. Gene fusion.. It is possible to engineering construct that consist of segment from two different genes. such construct are known as gene fusions.
- 13. Conti.. If the promoter that controls a coding sequence is removed, the coding sequence can be fused to different regularly region to place the gene under the control of different promoter alternatively. The promoter region can be fused to gene whose product is east to assay.
- 14. There are two types of gene fusion Fig.. . Operon fusion In operon fusion, a coading sequence that retain its own translation start site and signals is fused to the transcriptional signals of another gene.
- 15. Gene fusion Protein fusion In protein fusion, two coading sequence are fused with the result that they share the same transcriptional and traslational start sites and signals. Fig
- 16. Conti. Gene fusion are often used in studying gene regulation, especially if measuring the level of natural gene product is difficult and time consuming. The reulatery region of the gene of interest is fused to coding sequence for reporter gene, such as 硫- galactosidase or GFP. The reporter is then made under the condition that would trigger expression of the target gene. The expression of reporter is assay under variety of condition to determine how the gene of interest is regulated, where as protein fusion reveal translational cantrol.
- 18. Conti.. Gene fusion may also be used to test for the effect of regulatory genes. Mutation that effect regulatory genes are inroducted into cells carrying gene fusion, and expression is measured and compared to cell lacking the regulatory mutation. This allows the rapid screening of multiple regulatory genes that are suspected of controlling the target gene.