2.8 Experimental Techniques

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Tools and Techniques

 

Our ability to study and understand oncogenes goes hand in hand with advancements in genetic research techniques. Several strategies have been employed in an attempt to sequence genomes and subsequently identify various genes and their functions. With the completion of the Human Genome Project, the human genome has been successfully sequenced. As well, techniques such as chromatin immunoprecipitation (ChIP) and microarrays have been around for a long time and are continually being used in research today while next-generation sequencing methods such as RNA-Seq show a promising future for research models. 

 

 

Chromatin Immunoprecipitation (ChIP)

 

Chromatin Immunoprecipitation (ChIP) is an experimental technique that is used to explore the interactions between proteins (e.g. histones or transcription factors) and DNA. Monoclonal antibodies are used to selectively precipitate a protein of interest, which as a final result enriches the DNA fragments associated with the protein. In a ChIP experiment, DNA-binding proteins are first reversibly cross-linked to the nuclear DNA chromatin by treating cells with formaldehyde. The DNA chromatin with bound proteins is then sheared by sonication or subjected to mononuclease digestion, resulting in small fragments ranging between 200 - 600 base pairs. Combined with either agarose, sepharose, or magnetic beads, these DNA-protein complexes are then immunoprecipitated by antibodies specific to the protein of interest. As a result, the enriched sample predominantly consists of DNA-protein complexes that contain the targeted protein.  By reversing the crosslink interactions between the DNA fragment and protein, proteinase can be added to generate a final product of DNA fragments, which represent the genomic sites that are directly bound by the protein. The genomic sites associated with the protein of interest can be identified by sequencing the DNA fragments or by using microarrays. 

 

 

Microarrays

 

Representational oligonucleotide microarray analysis (ROMA) is a method which uses oligonucleotide probes to scan a genome of interest and determine gene copy numbers (3). This method can be useful in determining the presence of gene duplications implicated with the activity of some oncogenes in cancer.

Microarrays use a microchip coated with single-stranded nucleic acid sequences, which will hybridize with a complementary sequence in the sample of interest. They are a powerful set of assays designed to detect the presence of a given gene and its expression (2). Through this technique a variety of oncogenes and oncogeneic microRNAs (4) have been identified, and this technology remains a centerpiece for oncogene research today (1, 2). See Chapter 3, Experimental Approaches Section, for more details.

 

 

Oncogene Cloning

 

Another method for studying oncogenes is by oncogene cloning. This is an effective method for isolating genes without prior knowledge of its structure or sequence (5). The process of oncogene cloning begins with transformation of mouse NIH 3T3 fibroblast cells, which is achieved by transfecting fibroblasts with DNA derived from human tumor cells (6). The 3T3 mouse cells are cultured and a focus of transformed cells is detected growing within the untransformed cells. DNA from focus-derived cells is extracted and a second transfection carried out and a focus is again detected among the cultured cells (6). However, the efficiency of the first and second cycle transfers has been found to be quite low. DNA is extracted from these tumors, run on a Southern blot, and a probe for the Alu sequence is used to identify the portion of DNA carrying the oncogene on the original human DNA (5). Probes for Alu sequences are used because these sequences are highly abundant in the human genome, resulting in all genes being associated with one or multiple Alu sequences (5). After the second transfection, only human DNA and linked Alu sequences present in the DNA samples should contain the oncogene since it was extracted from the focus. Finally, genomic libraries can be constructed using DNA from secondary transformants and the oncogene cloned by detection with an Alu probe (5).

 

 

Next Generation Sequencing

 

RNA Sequencing (RNA-Seq) is just one of the more advanced methods currently being used for more precise annotation of genes. RNA-seq is a method that allows comparison of gene expression profiles with great sensitivity to transcripts which are expressed at a low level, making it particularly important in oncogenic mutations and tumour research (7). Strand specific RNA sequencing requires RNA extracted from tissues which is then reverse transcribed and amplified. Fragmented cDNA are ligated to adaptors and these transcripts are then sequenced. Their expression profiles are analyzed using the researcher's choice of sequencing technology (Illumina, SOLiD, or 454 technology) (8). Computational analysis can be done after sequencing to join all of the sequence fragments into a comprehensible representation of the transcripts expressed.

 

Coupled with the progressing technology of next-generation sequencing (NGS), binding sites detected by ChIP can be sequenced by investigators to create a genome wide profiling of specific protein-DNA interactions in a given cell type and experiment. In the case of this chapter, Chromatin-immunoprecipitation sequencing (ChIP-seq) is an indispensable tool that can be used to elucidate the gene regulation and epigenetic mechanisms associated with aberrant oncoproteins or tumor suppressor proteins. By determining which genes an oncoprotein or tumor suppressor protein directly regulates, researchers are able to determine the downstream functional role of the protein.

Moving forward from the discovery and identification of oncogenes and their role in cancer, subsequent sections of this chapter will examine the molecular function of oncogenes, highlight how several viruses use these genes or modified versions of them to their advantage and new frontiers in oncogene directed anti-cancer therapies.

 

Colony-Forming Assay

 

This method is used to identify cells which are expressing oncogenes from a heterogeneous population of cells. The primary idea of this assay is to differentiate potential oncogenes from a whole-genome DNA library derived from human cancer cells. The assay begins with the functional cloning of a human tumor-derived genomic library. The functional cloning of oncogenes is described above in great detail. After successful cloning of the DNA library into immortalized cells, The cell population is plated on a non-specific medium. Immortalized cells grow differently on plates than transformed cells do. transformed cells do not adhere to monolayer formation, whereas immortalized cells cannot grow beyond a monolayer. Cells that have been transformed, presumably due to the cloning of a potential oncogene, can be seen as a colony on the medium. These cells can be subsequently isolated and grown as a pure culture for further characterization of the cloned oncogene.

 

References:

1. Zender, L. et al. Identification and validation of oncogenes in liver cancer using an integrative oncogenomic approach. Cell 125, 1253-1267 (2006).

2. Segal, E., Friedman, N., Kaminski, N., Regev, A. & Koller, D. From signatures to models: understanding cancer using microarrays. Nature genetics 37, S38-S45 (2005).

3. Lucito, R. et al. Representational oligonucleotide microarray analysis: a high-resolution method to detect genome copy number variation. Genome research 13, 2291-2305 (2003).

4. Hammond, S.M. MicroRNAs as oncogenes. Current opinion in genetics & development 16, 4-9 (2006).

5. Weinberg, R. A. (2007). The Biology of Cancer. Garland Science.

6. Shimizu K, Nakatsu Y, Sekiguchi M, Hokamura K, Tanaka K, Terada M, Sugimura T. Molecular cloning of an activated human oncogene, homologous to v-raf, from primary stomach cancer. Proc. Natl. Acad. Sci. 82, 5641-5645 (1985).

7. Meyerson, M., Gabriel, S., & Getz, G. (2010). Advances in understanding cancer genomes through second-generation sequencing. Nature Reviews Genetics, 11(10): 685-696.

8. Martin, J.A. & Wang, Z. (2011). Next-generation transcriptome assembly. Nature Reviews Genetics, 12(10): 671-682.