Question

Those who inherit a mutant allele of the \(R B 1\) 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 \(R B 1\) allele, and you have cultured some osteosarcoma cells and obtained a cDNA clone of a normal human \(R B 1\) gene. A colleague sends you a research paper revealing that a strain of cancer-prone mice develops malignant tumors when injected with osteosarcoma cells, and you obtain these mice. Using these three resources, indicate which experiments you would perform to determine the following: (a) whether osteosarcoma cells carry two \(R B 1\) mutations, (b) whether osteosarcoma cells produce any pRB protein, (c) if the addition of a normal \(R B 1\) gene will change the cancercausing potential of osteosarcoma cells.

Short answer

Answer: The three experiments performed are: (a) Experiment A aims to determine if osteosarcoma cells carry two \(R B 1\) mutations by sequencing the \(R B 1\) genes in these cells. (b) Experiment B aims to determine if osteosarcoma cells produce any pRB protein using a Western blot. (c) Experiment C aims to determine if the addition of a normal \(R B 1\) gene can change the cancer-causing potential of osteosarcoma cells by comparing tumor growth and survival rates in cancer-prone mice injected with transfected and control osteosarcoma cells.

Step-by-step solution

Experiment A: Determine if osteosarcoma cells carry two \(R B 1\) mutations

To determine if osteosarcoma cells carry two \(R B 1\) mutations, we need to sequence the \(R B 1\) genes in these cells. First, extract the genomic DNA from the osteosarcoma cells. Then, amplify the \(R B 1\) gene region in the extracted genomic DNA using polymerase chain reaction (PCR). After that, sequence the PCR products to reveal any mutations in the \(R B 1\) gene.

Experiment B: Determine if osteosarcoma cells produce any pRB protein

To determine if osteosarcoma cells produce any pRB protein, we first need to prepare total protein extracts from the cells. Afterward, perform a Western blot using an antibody specific for pRB and appropriate secondary antibodies to detect its presence. A positive Western blot result would indicate the presence of pRB protein, whereas a negative result would indicate that osteosarcoma cells do not produce pRB protein.

Experiment C: Determine if a normal \(R B 1\) gene can change the cancer-causing potential of osteosarcoma cells

First, transfect the osteosarcoma cells with the normal human \(R B 1\) cDNA clone to create osteosarcoma cells that overexpress the normal \(R B 1\) gene. Verify the overexpression of \(R B 1\) through quantitative reverse transcription PCR (qRT-PCR) or Western blot. Next, inject both the transfected osteosarcoma cells and control (untransfected) osteosarcoma cells into separate groups of cancer-prone mice. Monitor tumor growth and survival rates in both groups of mice. A decrease in tumor growth or an increase in survival rate in mice injected with transfected osteosarcoma cells, compared to the control group, would suggest that adding a normal \(R B 1\) gene can change the cancer-causing potential of osteosarcoma cells.