Gus staining and reporter gene

Good And Informative... Gus Staining and Reporter Gene as Molecular Marker System...

1. Gus staining and reporter gene Pradeep Kumar MSc . (Ag.) Biotech.

2. A  selectable marker  is a  gene  introduced into a  cell, especially a  bacterium  or to cells in  culture, that confers a trait suitable for artificial selection . They are a type of reporter gene used in laboratory   microbiology , molecular biology, and genetic engineering to indicate the success of a  transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes; bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those bacterial colonies that can grow have successfully taken up and  expressed the introduced genetic material. Slectable marker

3. Normally the genes encoding resistance to antibiotics such as  ampicillin , chloroamphenicol , tetracycline or kanamycin, etc., are considered useful selectable markers for E.coli . Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional on the presence of external substrates . Positive selectable marker genes are defined as those that promote the growth of transformed tissue whereas negative selectable marker genes result in the death of the transformed tissue. continue

4. Non-selectable marker genes or reporter genes have been very important as partners to selectable marker gene systems. They have been used in co-transformation experiments to confirm transgenic events where escapes may be common. They have been used to improve transformation systems and the efficiency of recovering transgenic plants by allowing the visual detection of transformed tissues. Non-selectable marker genes or reporter genes may aid in the identification of the transformed cells. Non-selectable

5. Green fluorescent protein (GFP) has been particularly important in the development of these strategies, GFP has become a valuable tool for monitoring gene expression in field trials and for following pollen flow. Although destructive assays are needed to measure the activity of reporter genes such as GUS, As a reporter, luciferase (LUC) can be monitored in living tissue but this requires specialized detection equipment. Continue

6. In 1987, Richard Jefferson et.al , demonstrated the application of a new reporter gene system in transgenic plants. The reporter gene was the uid A gene of Eascherichia coli that encode the enzyme β - glucuronidase ( gus ). Uid A gene has become one of the most widely used reporter gene in plant molecular biology and microbiology. The most frequent use of the gus gene is as a reporter gene for promoter analysis, in both transient assays and in stable transformed plants. GUS as a reporter marker

7. It has been used to identified promoter elements involved in many aspects of regulation of gene expression such as tissue specific and developmental regulation hormonal regulation, response to wounding and photoregulation . The gus gene has also been used as a reporter gene in promoter trapping studies as a marker for the development of plant transformation procedure and to study the machanism of Agrobacterium tumefaciens mediated transformation. Cont……

8. Agrobacterium tumefaciens mediated transformation.

9. There are different possible glucuronides that can be used as substrates for the β- glucuronidase , depending on the type of detection needed (histochemical, spectrophotometrical , fluorimetrical). The most common substrate for GUS histochemical staining is 5-bromo-4-chloro-3-indolyl glucuronide (X- Gluc ): the product of the reaction is in this case a clear blue color. Other common substrates are p- nitrophenyl β -D- glucuronide for the spectrophotometric assay and 4-methylumbelliferyl β -D- glucouronide (MUG) for the fluorimetrical assay. Histostaining for Tissue Expression Pattern of Promoter-driven GUS Activity in Arabidopsis

10. GUS-system in Arabidopsis thaliana Structure of GUS

11. Harvest tissue and place in cold 90% Acetone on ice. This should stay on ice until all samples are harvested. For sample containers, Eppendorf tubes and glass scintillation vials work well. When all samples are harvested, place at room temperature (RT) for 20 min. Remove acetone from the samples, and add staining buffer on ice. Add X- Gluc to the staining buffer to a final concentration of 2 mM from a 100 mM stock solution of X- Gluc in DMF- this must be kept in the dark at -20 °C. Remove staining buffer from samples and add staining buffer with X- Gluc on ice. Infiltrate the samples under vacuum, on ice, for 15 to 20 min. Release the vacuum slowly and verify that all the samples sink. If they don't, infiltrate again until they all sink to the bottom when the vacuum is released. Procedure

12. Incubate at 37 °C (I usually do it for 2 h for strong promotors and up to overnight for weak promotors . It is not advisable from my experience to go too long (over two days) as the tissue seems to begin deteriorating during long incubations. Remove samples from incubator and remove staining buffer. Go through ethanol series from 10%, 30%, 50%, 70% (you may heat the sample to 60 °C to get rid of chloroplasts), to 95% (avoid light); 30 min each step and then finally 100%. You may store at 4 °C for up to a month, seal well. Go to embedding procedure, or observe directly under dissecting or light microscope. To mount, simply apply a few drops of water to the samples. Cont….

13. β- Glucuronidase (GUS) is a very versatile reporter of gene expression that is frequently used in plant molecular biology. The diverse applications of the GUS gene fusion systems (Gallagher, 1992) are based on the detection of the enzymatic activity of GUS in protein extracts or in tissues using fluorometric and histochemical assays respectively. The histochemical assay has also been used for sub-cellular localization of GUS fusion proteins, e.g. for the nuclear targeting of important regulatory proteins (for review see Raikehl , 1994). A novel application of the GUS reporter was demonstrated for protein fusions with the A. thaliana ATHSFI heat shock transcription factor (Lee et al., 1995) using a fluorescence activity staining protocol following gel electrophoresis. GUS gene fusion system

14. A useful feature of GUS is that it can be fused with other proteins. Ex- GUS fusions with selectable marker genes such as nptII allow the visualization of transformation in addition to selection. GUS expression was used as a reporter to help detect transformation events in tissue culture during the production of a number of plant lines approved for commercialization . Continue

15. Heat stress treatment- Approximately 2 g plant tissue (leaves) are incubated at 37°C for 30 min. to two hours in an shaking water bath in SIB-puffer. Control tissue is incubated in a buffer at 25 °C. Cell disruption- Tissue is washed in cold water, blotted dry with paper towels and subsequently grind (0.3 g) in 100 µl extraction buffer in a 1.5 ml cup. Centrifugation- Spin down in a microfuge for 10 minutes at 12000 rpm at 4 °C and collect the supernatant in a fresh cup. Native page - Pour 5% or 7% polyacrylamide gel in Tris pH 8.8 buffer, apply Tris / Glycine (6 g/ 15 g/l ) running buffer, load native protein samples up to 50 µg/lane, and molecular weight standards (Pharmacia) and run over night at 70 V . Steps in procedure for GUS activity staining in gels

16. GUS activity staining (fluorescence method). Rinse gel with 50 mM Na-Phosphate buffer (PH 7) and incubate immediately in a solution containing 0.5 mM MUG in phosphate buffer for 10 min, 37°C. Visualization, recording. Following electrophoresis the GUS fusion proteins are visualized immediately by fluorescence of the generated MU on a UV transilluminator at 360 nm. The bands can be recordered photographically using Polaroid 667 film. Continue

17. The expression and regulation of a rice Glycine –rich cell wall protein gene, osgrp1 , transgenic rice plants were regulated that contain the osgrp1 promoter or its 5’ deletions fused with the bacterial β - glucuronidase (GUS) receptor gene. In root, of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions and no gus expression was detectable in apical meristem and the mature region. In shoot, apices Gus activity was detected only in those leaf cells which were starting to expend and differentiate and little Gus activity was expressed in mature leaves or mature parts of developing leaves. Gus expression was not detected in the apical meristem and the young meristematic leaf primordia . Gus activity was highly expressed in the young stem tissue particularly in the developing vascular bundles and epidermis. Application of gus gene in plants

18. The expression of the osgrp1 gene is closely associated with cell elongation expansion during the post-meiotic cell differentation process. The osgrp1 gus gene was also expressed in response to wounding and down regulated by water stress condition in the elongation region of roots. Cont.. Promoter deletion analysis indicates that both positive and negative mechanism are involved in regulating the specific expression patterns .

19. GUS was expressed in transgenic yeast on a Multiplecopy vector under the control of the alcohol dehydrogenase 1 ( ADH1) promoter. GUS as a reporter for targeting proteins into different subcellular compartments in vivo, we fused the presequence of the mitochondrial tryptophanyl tRNAsynthetase gene (MSW) to the amino terminus of GUS. Enzyme is stable in yeast and its activity may be monitored by very sensitive colorimetric or fluorometric methods in extracts, or by the histochemical reagent 5-bromo-4-chloro-3-indolylglucuronide (X- Gluc ) on plates. Application of the β - glucuronidase gene fusion system to Saccharomyces cerevisiae

20. GUS expression was used as a reporter to help detect transformation events in tissue culture during the production of a number of plant lines approved for commercialization. These lines include Bollgard II® cotton, the glyphosate resistant sugar beet line GTSB77 (variety InVigorTM ), papaya line 55-1, three soybean lines with modified fatty acid content (G94-1, G94-19, G168) and two PPT tolerant soybean lines (W62 and W68) . With 91 records, GUS is the most frequently listed reporter gene in the US field trials database in 2001 and 2002. Other application

21. GUS enzyme is very stable within plants and is non-toxic when expressed at high levels. A useful feature of GUS is that it can be fused with other proteins i.e. GUS fusions with selectable marker genes such as npt II allow the visualization of transformation in addition to selection. Humans and animals are continuously exposed to GUS from bacteria residing in their intestinal tracts and from non-transgenic food sources without harmful effects; therefore, the low level of GUS protein from genetically modified plants is not a concern with regard to toxicity or allergenicity . Advantage

22. The major drawback with the use of GUS as a reporter is that the assays are destructive to the plant cells. Reporter assays in a given system can be readily adapted to almost any gene of interest, without the need to develop separate assays for individual gene products, which are sometimes very difficult or laborious. DISADVANTAGE

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