Reporter gene

Source: Wikipedia, the free encyclopedia.
A diagram of a how a reporter gene is used to study a regulatory sequence.

In

selectable markers
. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population.

Common reporter genes

To introduce a reporter gene into an organism, scientists place the reporter gene and the gene of interest in the same

viruses, this is known as a viral vector. It is important to use a reporter gene that is not natively expressed in the cell or organism under study, since the expression of the reporter is being used as a marker for successful uptake of the gene of interest.[1]

Commonly used reporter genes that induce visually identifiable characteristics usually involve

dsRed [fr].[3][4][5][6][7] The GUS gene has been commonly used in plants but luciferase and GFP are becoming more common.[8][9]

A common reporter in bacteria is the

beta-galactosidase.[10] This enzyme causes bacteria expressing the gene to appear blue when grown on a medium that contains the substrate analog X-gal. An example of a selectable marker which is also a reporter in bacteria is the chloramphenicol acetyltransferase (CAT) gene, which confers resistance to the antibiotic chloramphenicol.[11]

Gene name Gene product Assay Ref.
lacZ
β-galactosidase
 Enzyme assay, Histochemical [10]
cat Chloramphenicol acetyltransferase Chloramphenicol acetylation [11]
gfp Green fluorescent protein Fluorescent [3]
rfp Red fluorescent protein Microscopical, Spectrophotometry [12]
luc Luciferase enzyme Bioluminescence [4]

Transformation and transfection assays

Many methods of

promoter (DNA regions that initiates gene transcription) independent from that of the introduced gene of interest; the reporter gene can be expressed constitutively (that is, it is "always on") or inducibly with an external intervention such as the introduction of Isopropyl β-D-1-thiogalactopyranoside (IPTG) in the β-galactosidase system.[10] As a result, the reporter gene's expression is independent of the gene of interest's expression, which is an advantage when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access.[1]

In the case of selectable-marker reporters such as CAT, the transfected population of bacteria can be grown on a substrate that contains chloramphenicol. Only those cells that have successfully taken up the construct containing the CAT gene will survive and multiply under these conditions.[11]

Gene expression assays

Reporter genes can be used to assay for the expression of a gene of interest that is normally difficult to quantitatively assay.[1] Reporter genes can produce a protein that has little obvious or immediate effect on the cell culture or organism. They are ideally not present in the native genome to be able to isolate reporter gene expression as a result of the gene of interest's expression.[1][15]

To activate reporter genes, they can be expressed

mRNA is then translated into protein. It is important that both proteins be able to properly fold into their active conformations and interact with their substrates despite being fused. In building the DNA construct, a segment of DNA coding for a flexible polypeptide linker region is usually included so that the reporter and the gene product will only minimally interfere with one another.[17][18] Reporter genes can also be expressed by induction during growth. In these cases, trans-acting elements, such as transcription factors are used to express the reporter gene.[19][20]

Reporter gene assay have been increasingly used in high throughput screening (HTS) to identify small molecule inhibitors and activators of protein targets and pathways for drug discovery and chemical biology. Because the reporter enzymes themselves (e.g. firefly luciferase) can be direct targets of small molecules and confound the interpretation of HTS data, novel coincidence reporter designs incorporating artifact suppression have been developed.[21][22]

Promoter assays

Reporter genes can be used to assay for the activity of a particular promoter in a cell or organism.[23] In this case there is no separate "gene of interest"; the reporter gene is simply placed under the control of the target promoter and the reporter gene product's activity is quantitatively measured. The results are normally reported relative to the activity under a "consensus" promoter known to induce strong gene expression.[24]

Further uses

A more complex use of reporter genes on a large scale is in two-hybrid screening, which aims to identify proteins that natively interact with one another in vivo.[25]

See also

References

  1. ^
    ISBN 978-90-481-3261-4
  2. PMID 11740505
    .
  3. ^
    PMID 15640280
    .
  4. ^
    PMID 20439408
    .
  5. PMID 11737785
    .
  6. S2CID 9184712
    .
  7. PMID 12799012
    .
  8. PMID 8563799
  9. PMID 17597079
    .
  10. ^
    PMID 20439410
    .
  11. ^
    PMID 20439409
    .
  12. S2CID 12713372
    .
  13. PMID 1943786
  14. PMID 6345791
    .
  15. ^ Archived at Ghostarchive and the Wayback Machine: Promega Corporation, Promega Corporation (October 22, 2014). "Introduction to Reporter Gene Assays". YouTube. Retrieved March 21, 2020.
  16. ISBN 978-3-319-27656-4
    .
  17. PMID 22484672
    .
  18. PMID 23026637
    .
  19. S2CID 91744525
    , retrieved 2019-12-16
  20. PMID 31366153
    .
  21. PMID 23018994
    .
  22. S2CID 20139739
    .
  23. PMID 27547572
    .
  24. PMID 23436354
    .
  25. PMID 19582228
    .

External links
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