Those who inherit a mutant allele of the RB1 tumor-suppressor gene are at risk for developing a bone cancer called osteosarcoma. You suspect that in these cases, osteosarcoma requires a mutation in the second RB1 allele, and you have cultured some osteosarcoma cells and obtained a cDNA clone of a normal human RB1 gene. A colleague sends you a research paper revealing that a strain of cancer-prone mice develop malignant tumors when injected with osteosarcoma cells, and you obtain these mice. Using these three resources, what experiments would you perform to determine (a) whether osteosarcoma cells carry two RB1 mutations, (b) whether osteosarcoma cells produce any pRB protein, and (c) if the addition of a normal RB1 gene will change the cancer-causing potential of osteosarcoma cells?

Verified step by step guidance

1. Design an experiment to sequence the RB1 gene from the osteosarcoma cells to identify mutations in both alleles. Use PCR to amplify the RB1 gene, followed by sequencing to compare with the normal RB1 sequence.
2. Perform a Western blot analysis to detect the presence of pRB protein in osteosarcoma cells. Extract proteins from the cells, run them on an SDS-PAGE gel, and use an antibody specific to pRB to determine if the protein is expressed.
3. Introduce the normal RB1 cDNA clone into osteosarcoma cells using a suitable vector for gene expression. Use a control group of osteosarcoma cells without the RB1 cDNA for comparison.
4. Inject both the modified osteosarcoma cells (with normal RB1) and the control cells into the cancer-prone mice. Monitor the development of tumors in both groups of mice over time.
5. Analyze the tumor development results to determine if the introduction of the normal RB1 gene reduces the cancer-causing potential of the osteosarcoma cells compared to the control group.

Key Concepts

RB1 Tumor-Suppressor Gene

The RB1 gene encodes the retinoblastoma protein (pRB), which is crucial for regulating the cell cycle and preventing uncontrolled cell growth. Mutations in this gene can lead to the loss of its tumor-suppressing function, increasing the risk of cancers such as osteosarcoma. Understanding the role of RB1 in cell cycle regulation is essential for analyzing how mutations contribute to tumorigenesis.

cDNA and Gene Expression Analysis

cDNA (complementary DNA) is synthesized from mRNA and is used to study gene expression. By comparing the cDNA of osteosarcoma cells to that of normal cells, researchers can determine whether the RB1 gene is expressed and if the pRB protein is produced. This analysis is vital for assessing the functional status of the RB1 gene in cancer cells.

Experimental Models in Cancer Research

Using animal models, such as cancer-prone mice, allows researchers to study the effects of specific genetic mutations on tumor development. By injecting these mice with osteosarcoma cells, one can observe the tumorigenic potential and the impact of introducing a normal RB1 gene. This approach helps elucidate the mechanisms of cancer progression and the potential for gene therapy.