reporter gene assays by Tahura Mariyam

this presentation is about reporter gene essay, its types, blue white screening and its application, Antibiotic resistance gene and Herbicide resistance markers

1. TOPIC- R eporter G ene A ssay PRESENTED BY: TAHURA MARIYAM MSc. MICROBIOLOGY (Sem -II) P.ID: 19MSCMB009 PRENENTED TO: Dr. Gurudayal Ram (Assistant prof. Sr. Grade) DEPARTMENT OF INDUSTRIAL MICROBIOLOGY JACOB INSTITUTE OF BIOTECHNOLOGY AND BIO-ENGINEERING SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE,TECHNOLOGY, AND SCIENCES, PRAYAGRAJ

2. CONTENTS Reporter gene essay An ideal reporter gene Types of reporter gene 1- Scorable reporter gene: GFP, variants of GFP GUS, Luciferase, Beta-galactosidase Common reporter gene Green Fluorescent Protein (GFP) Luciferase GUS assay Blue white screen 2- Selectable Reporter gene Antibiotic resistance gene Herbicide resistance markers Reporter gene for functional genomics Gene expression assays Transfection assays Applications of reporter gene assay

3. What are reporter genes? Gene whose products are easily detected or monitored A gene that is used to `tag' another gene or DNA sequence of interest for: Identifying whether a certain gene has been taken up by cell Measurement of gene expression

4. An ideal reporter gene Easily quantifiable Relatively rapid degradation of the enzyme Lack of endogenous activity in the concerned cell Should not be toxic to cells Assay should be sensitive and reliable

5. Types of Reporter gene Scorable reporter genes Expression of this results in quantifiable phenotype Easily detected through highly sensitive enzyme assays 2. Selectable reporter genes Expression of resistance to a toxin Selection of transformants from non transformants in growth media containing selective agent.

6. 1- Scorable reporter gene :- Green Fluorescent Protein (GFP ) Derived from jellyfish Aequorea Victoria. Formed by nucleophilic reaction between C- ter of S with N- ter of G, formed imidazoline heterocyclic ring which oxidise with Y to yield florescence Variants of GFP :- Yellow Fluorescent Protein Formed by mutation of Thr 203 residue to tyrosine Blue Fluorescent Protein Modification of tyr66 to his

7. Cyan Fluorescent Protein Modification of tyr66 to tryptophan Red fluorescent protein Derived from Discosoma striata (Ds Red) Alternation of >30 amino acid to yield RFP-1

8. GUS Derived from E. Coli uid A gene code for 12- beta- glucuronidase enzyme Enzymatic cleavage of X- Gluc (5-bromo-4-chloro-3-indolyl beta-D- glucuronide) undergoes an oxidative dimerization to yield an indigo blue precipitate GUS assay (using β- glucuronidase ) is an excellent method for detecting a single cell by staining it blue without using any complicated equipment. The drawback is that the cells are killed in the process. It is particularly common in plant science.

9. Luciferase Bacterial luciferase : Vibrio harveyi ( luxA / luxB genes) Firefly ( Photinus pyralis ) luciferase ( luc gene ) β- galactosidase ( LacZ ) Derived from : E. coli catalyzes the hydrolysis of X-Gal producing a blue precipitate

10. Common reporter genes (Scorable reporter gene) Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Gene name Gene product Assay lacZ β-galactosidase Enzyme assay, Histochemical cat Chloramphenicol acetyltransferase Chloramphenicol acetylation gfp Green fluorescent protein Fluorescent rfp Red fluorescent protein Microscopical, Spectrophotometry luc Luciferase enzyme Bioluminescence

11. Scorable reporter genes Green fluorescent protein (GFP) Green fluorescent protein (GFP) causes cells that express it to glow green under UV light. A specialized microscope is required to see individual cells. Yellow and red versions are also available, allowing the investigation of multiple genes at once. It is commonly used to measure gene expression . From jellyfish Aquorea victoria , glows in blue light 395nm giving green fluorescence (510nm ) allows non-destructive imaging of plants and sub cellular localization of GFP by microscopy GFP is a small protein of 238 amino acids . Different variants like EGFP, Red GFP, EYFP, etc available . Does not require any substrate, can be detected directly Can be detected invivo (non destructively) by using fluorescence microscope

12. Luciferase Luciferase as a laboratory reagent often refers to P. pyralis luciferase, although recombinant luciferases from several other species of fireflies are also commercially available. The luciferase enzyme catalyzes a reaction with its substrate (usually luciferin) to produce yellow-green or blue light, depending on the luciferase gene. Since light excitation is not needed for luciferase bioluminescence, there is minimal autofluorescence and thus virtually background-free fluorescence .

13. pGL4 Luciferase Reporter Vectors Encoding Firefly and Renilla Luciferase Because vectors are used to deliver the reporter gene to host cells, regulatory sequences such as transcription factor-binding sites and promoter modules within the vector backbone can lead to high background and anomalous responses. This is a common issue for mammalian reporter vectors, including the pGL3 Luciferase Reporter Vectors. Our scientists applied the successful "cleaning" strategy described for reporter genes to the entire pGL3 Vector backbone, removing cryptic regulatory sequences wherever possible, while maintaining reporter functionality.

14. A comparison of the sensitivity of NanoLuc , firefly and Renilla luciferase assays

15. Bioluminescent Reporter Genes GUS assay (using β- glucuronidase ) is an excellent method for detecting a single cell by staining it blue without using any complicated equipment. The drawback is that the cells are killed in the process. It is particularly common in plant science GUS is probably the most widely used reporter gene in plants  low endogenous activity in plant stable enzyme which hydrolyses wide range of ß-glucuronides. easily assayed for histochemical analysis, using X- gluc (5-bromo, 4- chloro , 3-indolyl ß–glucuronide). After cleavage, oxidation of the indole derivative causes dimerisation and the production of an insoluble indigo dye

16. BETA GLUCURONIDASE (GUS) ASSAY

17. Blue-white screen Blue-white screen is used in both bacteria and eukaryotic cells. The bacterial lacZ gene encodes a β-galactosidase enzyme. When media containing certain galactosides (e.g., X-gal) is added, cells expressing the gene convert the X-gal to a blue product and can be seen with the naked eye . Blue-White Screen

18. 2- Selectable reporter gene i) Antibiotic Resistance Genes Neomycin phosphotransferase II ( npt II gene) Derived from the transposon Tn5 code foraminoglycoside 3` phosphotransferase Resistance to the antibiotic kanamycin neomycin by phosphorylation Hygromycin phosphotransferase ( hpt gene) Derives from E. coli Resistant against hygromycin by phosphorylation ii) Herbicide Resistance Markers Phosphinothricin acetytransferase (pat/bar gene) Derived from Streptomyces hygroscopicus Converts herbicides into acetylated forms Resistant against Bialophos , phosphinothricin and glufosinate Enolpyruvylshikimate phosphate synthase ( epsps / aroA genes) Derived from Agrobacterium sp CP4 Resistance against glyphosate which blocks the activity of EPSP synthase, a key enzyme involved in the biosynthesis of aromatic amino acid

19. Bromoxynil nitrilase ( bxn gene) Derived from Klebsiella pneumoniae The herbicide bromoxynil inhibits photosynthesis (photosystem II) Encode a specific nitrilase that converts bromoxynil to its primary metabolite 3,5-dibromo-4-hydroxybenzoic acid iii) Reporter gene for functional genomics Identify a promoter, to study the expression pattern and strength of the promoter Reporter gene is simply placed under the control of the target promoter a ) Gene expression assays Reporter is directly attached to the gene of interest to create a gene fusion The two genes are under the same promoter elements and are transcribed and then translated into protein b) Transformation and transfection assays • Reporter genes expressed under their own promoter independent from that of the introduced gene of interest • Reporter gene can be expressed constitutively or inducibly

20. a) Gene expression assays Reporter gene assays are invaluable for studying regulation of gene expression, both by cis-acting factors (gene regulatory elements) and trans-acting factors (transcription factors or exogenous regulators). Furthermore, reporter gene systems enable the use of pathway-specific, tissue-specific, or developmentally regulated gene promoters as biomarkers for specific events processes. In these assays, the detectable reporter gene acts as a surrogate for the coding region of the gene under study. The reporter gene construct contains one or more gene regulatory elements to be analyzed, the sequence for the reporter gene, and the sequences required for the transcription of functional mRNA. Upon introduction of the reporter construct into cells, expression levels of the reporter gene are monitored through a direct assay of the reporter proteins enzymatic activity.

21. b) Transfection assays In contrast to selectable markers, which protect an organism from a selective agent that would normally kill it or prevent its growth, reporter genes used for screening transfectants make the cells containing the reporter gene visually identifiable. Reporter genes used in this way are normally expressed under their own promoter independent from that of the introduced gene of interest, allowing the screening of successfully transfected cells even when the gene of interest is only expressed under certain specific conditions or in tissues that are difficult to access. Reporter genes can also serve as controls for transfection. For example, transfection efficiencies between different experiments can be normalized by comparing the expression levels of a reporter gene used in all of the experiments.

22. Transformation and gene expression assay Transformation Assay TaqMan genotyping assays

23. Applications of reporter gene The conventional use of reporter genes is largely to analyze gene expression and dissect the function of cis-acting genetic elements such as promoters and enhancers (so-called "promoter bashing"). In typical experiments, deletions or mutations are made in a promoter region, and their effects on coupled expression of a reporter gene are quantitated. However, reporter genes also can be used to study other cellular events, including events that are not related to gene expression such as cell health and signaling pathways . Normalize for Changes in Cell Physiology Monitor RNA Interference

24. Examine Nuclear Receptors Bioluminescent reporter genes can also characterize nuclear receptors, a class of ligand-regulated transcription factors that sense the presence of steroids and other molecules inside the cell. Nuclear receptors typically reside in the cytoplasm and are often complexed with associated regulatory proteins. Ligand binding triggers translocation into the nucleus, where the receptors bind specific response elements via the DNA-binding domain, leading to upregulation of the adjacent gene. Bioluminescent reporters can be harnessed to identify and characterize nuclear receptor agonists, antagonists, co-repressors and co-activators using a universal receptor assay. The universal nuclear reporter assay can be thought of as a "one-hybrid" assay, where the ligand-binding domain (LBD) of a nuclear receptor is fused to yeast GAL4 transcription factor and when a ligand binds to the nuclear receptor, firefly luciferase is expressed

25. REFERENCE https://www.promega.in/resources/guides/cell-biology/bioluminescent-reporters/ https://info.gbiosciences.com/blog/role-of-reporter-genes-to-assay-for-transcription-factors-more https://en.m.wikipedia.org/wiki/Reporter_gene