A new transcription unit was discovered while working with transfected murine BAC clones, because a novel spot appeared on a 2-dimensional protein gel. Through a process of expression subcloning from the BAC clone, the transcription unit that generated the novel peptide was located. This finding was back validated by sequencing the protein contained in the 2-D gel piece using N-terminal Edman degradation and mass spectroscopy (Rosenfeld, Capdevielle, Guillemot, and Ferrara, 1992; Hellman, Wernstedt, Gonez, and Heldin, 1995).
This transcription unit happens to be encoded within a multi-gene locus that is coordinately regulated in a tissue-specific and developmental manner. Since the laboratory is already heavily invested in characterizing the gene regulatory mechanisms that control this locus, and the downstream roles of the gene products, understanding the expression pattern of this novel gene may be important to ongoing research efforts.
Determination of mRNA patterns of expression
In addition to gearing up for producing this protein for both polyclonal and monoclonal antibody production, a substantial amount of information can be obtained by analyzing the tissue expression pattern(s) of the RNA transcript that encodes this protein. Commercial RNA preparations are available that allow rapid screening of mouse tissues (for example see ClonTech, 2011), including embryonic stages, using RT-PCR. If the gene is suspected of being expressed at low levels then a real-time PCR approach will be utilized. Additional controls will be needed, including the quantification of three to five housekeeping gene targets to control for any variations in reverse transcriptase reaction efficiency between tissue samples (Xu, Ma, Cui, Li, Ning, and Wang, 2010). In addition, yeast RNA will be used to control for any sample contamination issues that could arise (negative control).
Once the tissues and developmental stages expressing the transcript has been established using RT-PCR, then the location within mouse embryos and tissues can be further refined using in situ hybridization (Yan et al., 2010; Carter, Fletcher, and Thompson, 2010). Tissues suspected to be expressing the transcript are fixed in paraformaldehyde and embedded in paraffin wax. Microtome slices are prepared and allowed to dry on glass slides. After removing the wax, the tissue slices are blocked with a solution containing yeast RNA and Denhardt's reagent (milk protein, carbohydrates, and vinyl polymers), and then incubated with the fluorescently-tagged RNA probe. After several washings, RNAse A treatment, and H&E counter-staining, the location of the probe within the tissue is visualized and captured digitally using fluorescent and light microscopy. Should the fluorescent probe fail to provide a good signal-to-noise ratio, radioactive RNA probes will be used.
Determination of protein patterns of expression
The development of an antibody probe can be subcontracted out for a sizeable fee, or bacterial-expressed peptides can be injected into rabbits and mice in-house in an effort to generate polyclonal (Cooper, and Paterson, 2008) or monoclonal (Yokoyama, 2006) antibodies, respectively. Confirmation of the specificity of the antibody can be accomplished by probing Western blots containing protein extracts from known positive and negative controls (Gallagher, Winston, Fuller, and Hurrell, 2008). A positive control can be created by stably transfecting an expression construct into the mouse fibroblast 3T3 cell line together with a selectable marker like the neomycin resistance gene, expanding colonies in G418-containing medium, and preparing protein extracts from the expanded clones (Mortensen and Kingston, 2009). Bacterial protein extracts can serve as a negative control, unless prior RT-PCR screening of available murine cell lines revealed one or more are non-expressing. Should background problems occur the antibody will be affinity-purified. Should any of the antibodies prove to work well on Western blots, protein extracts from tissues expressing and not expressing the transcript will be screened by Western blot.
The figure to the right shows a dilution series determining the optimal antibody concentration for low background. Lanes are 1/50, 1/100, 1/200, 1/400, 1/800, 1/1600, 1/3200, 1/6400 from left to right. The molecular weight ladder on the right goes from 200, 116, 97, 66, 43, 24, and 18 kDa (Gallagher, Winston, Fuller, and Hurrell, 2008).
If the antibody performs well in the Western blotting of murine tissues, then it will be used to probe tissue sections for the presence of the novel protein using fluorescent microscopy. Tissue sections are blocked with milk protein, washed, incubated with the antibody in blocking solution, and then washed repeatedly. The tissue sections are then incubated with a secondary antibody conjugated to a fluorescent chromophore or horseradish peroxidase. Sections will be counterstained with H&E or other appropriate stains or primary antibodies. Visualization is by fluorescent or light microscopy, respectively, after incubating with fluorescent chromogen-tagged secondary antibodies (Daneshtalab, Dore, and Smeda, 2010).
Should the antibody perform well in Western blots and as an immunohistochemistry probe, then the subcellular location of the novel protein by confocal laser scanning microscopy will be attempted (Smith, 2008). Tissues are fixed in 2-4% formaldehyde, embedded in paraffin wax, and mounted on glass slides. After removing the wax, sections are blocked, and then incubated with the primary antibody. After several washes the tissue section is incubated with the fluorescently-tagged secondary antibody, washed, and then visualized by excitation using a laser operating in the visible or ultraviolet wavelength range. Sections will be counterstained for common cytoplasmic or nuclear components, such as laminin, nucleoporin, histone, and microtubules to help identify common subcellular structures associated with the novel protein.
The figure to the right shows staining for three different transcription factors in the drosophila embryo (panels A and B) visualized using optical fluorescent microscopy. Panel C. shows staining of tubulin (green), mitochondria (red), and DNA (DAPI) and visualized using 20, 0.3 um optical sections and a 100X objective lens. Panel D. shows GFP staining of the Golgi complex as part of a photobleaching experiment (FRAP) in living cells (Smith, 2008).
If the novel protein is associated with a subcellular organelle or structure, this information can be compared with the results of protein sequence homology prediction software. A number of online and commercial protein homology databases and comparison tools are available, including the Conserved Domain Database (CDD) accessed through NCBI (Marchler-Bauer, Lu, Anderson, Chitsaz, Derbyshire, Deweese-Scott, et al., 2010). For example, if confocal imaging data indicates a nuclear location then the presences of a conserved DNA-binding domain encoded in the novel protein would suggest the possibility that it function as a transcription factor. This hypothesis could be tested by mutating the DNA sequence encoding the DNA-binding domain to generate non-synonymous changes in amino acid sequence. Expressing the mutated novel protein fused to a fluorescent protein (GFP for example) will facilitate visualization of disrupted binding (Kahana, and Silver, 2001).
Determining the expression pattern of a protein can provide a considerable amount of information about what its function may be. For example, expression in the subventricular zone of the forebrain during embryonic day 10.5 would suggest a role in neurogenesis, whereas expression four days later may suggest a role in gliogenesis (Battiste, Helms, Kim, Savage, Lagace, Mandyam, et al., 2006). If the protein is found to be confined to the nucleus then it may function as a transcription factor or nuclear envelope protein. This process of identifying the temporal and spatial expression patterns of the mRNA and protein is therefore part of a long-term goal that eventually leads to characterizing its in vivo functional role.
Battiste, J., Helms, A.W., Kim, E.J., Savage, T.K., Lagace, D.C., Mandyam, C.D. et al. (2006). Ascl1 defines sequentially generated lineage-restricted neuronal and oligodendrocyte precursor cells in the spinal cord. Development, 134, 285-293.
Carter, B.S., Fletcher, J.S., & Thompson, R.C. (2010). Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription. Methods, 52, 322-331.
ClonTech. (2011). Total RNA Master Panels. Retrieved February 6, 2011 from http://www.clontech.com/products/detail.asp?product_id=10600&tabno=2
Cooper, H.M. And Paterson, Y. (2008). Production of polyclonal antisera. In F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl (Eds.), Current Protocols in Molecular Biology (Unit 14.11). Wiley Online Library. Retrieved February 6, 2011 from http://www.currentprotocols.com/protocol/mb1112
Daneshtalab, N., Dore, J.J., and Smeda, J.S. (2010). Troubleshooting tissue specificity and antibody selection: Procedures in immunohistochemical studies. Journal of Pharmacological and Toxicological Methods, 61, 127-135.
Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G.R. (2008). Immunoblotting and Immunodetection. In F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl (Eds.), Current Protocols in Molecular Biology (Unit 10.8). Wiley Online Library. Retrieved February 6, 2011 from http://www.currentprotocols.com/protocol/mb1008
Hellman, U., Wernstedt, C., Gonez, J., and Heldin, C. (1995). Improvement of an "In-Gel" digestion procedure for the micropreparation of internal protein fragments for amino acid sequencing. Analytical Biochemistry, 224, 451-455.
Kahana, J.A. And Silver, P.A. (2001). Use of the A. Victoria Green Fluorescent Protein to study protein dynamics in vivo. In F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith and K. Struhl (Eds.), Current Protocols in Molecular Biology (Unit 10.8). Wiley Online Library. Retrieved February 6, 2011 from http://onlinelibrary.wiley.com/doi/10.1002/0471142727.mb0907cs34/pdf
Marchler-Bauer, A., Lu, S., Anderson, J.B., Chitsaz, F., Derbyshire, M.K., Deweese-Scott, C. et al., (2010). CDD: A Conserved Domain Database for…