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Production of proto-oncogene expressing control tissues for in situ hybridization and immunohistochemical studies
M D Johnson1, B W Davis, N T Wetherall
1Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232.
Abstract:
There is increasing interest in cellular localization of protooncogene, growth factor, and growth factor receptor gene expression in developing or neoplastic human tissue. Establishment of nucleic acid in situ hybridization (ISH) and immunohistochemical (IH) analyses of these genes, however, is compromised by the paucity of available control tissues which have been shown to express these cancer associated genes. The present study evaluated whether proto-oncogene or growth factor gene expressing human control tissues suitable for ISH and IH studies could be produced from malignant cell lines implanted into irradiated nude mice. Tumors rapidly fixed in parafomaldehyde 2 to 4 weeks after xenograft were found optimal for in situ hybridization and immunohistochemistry. Northern blots of frozen tumors confirmed the expression of specific proto-oncogenes in the tissue and the specificity of cDNA and cRNA probes for those transcripts. Production of control tissue from xenografted cell lines should facilitate establishment and long term quality control of in situ hybridization and immunohistochemical analysis of proto-oncogene or growth factor gene expression in laboratories with limited experience in these techniques.
Insights
Researchers developed a method to create human control tissues expressing cancer-associated genes. This technique uses xenografts in mice, providing valuable resources for gene expression studies like in situ hybridization and immunohistochemistry.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Growing interest in cellular localization of protooncogene, growth factor, and growth factor receptor gene expression in human tissues.
- Lack of suitable control tissues hinders nucleic acid in situ hybridization (ISH) and immunohistochemical (IH) analyses for these genes.
Purpose of the Study:
- To evaluate the feasibility of producing human control tissues expressing proto-oncogene or growth factor genes using xenografts.
- To establish a reliable method for generating control tissues for ISH and IH studies.
Main Methods:
- Implantation of malignant cell lines into irradiated nude mice to create xenografts.
- Rapid fixation of xenograft tumors in paraformaldehyde.
- Confirmation of gene expression using Northern blots and probe specificity testing.
Main Results:
- Tumors harvested 2 to 4 weeks after xenografting were optimal for ISH and IH.
- Northern blots confirmed expression of specific proto-oncogenes in xenografted tumors.
- Validated probe specificity for target transcripts.
Conclusions:
- Xenografting malignant cell lines in nude mice provides a viable method for producing human control tissues.
- This approach facilitates the establishment and quality control of ISH and IH analyses for cancer-associated genes.
- Beneficial for laboratories with limited experience in these techniques.