Those who inherit a mutant allele of the \(R B 1\) 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 \(R B I\) 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 \(R B 1\) mutations, (b) whether osteosarcoma cells produce any \(\mathrm{pRB}\) protein, and (c) if the addition of a normal \(R B 1\) gene will change the cancer-causing notential of astensarcama cells?

Step by step solution

01

(a) Determine if osteosarcoma cells carry two RB1 mutations

1. Extract DNA from the cultured osteosarcoma cells. 2. Amplify the RB1 gene region by PCR. 3. Perform DNA sequencing on the amplified region to determine the sequence of both RB1 alleles. 4. Compare the obtained sequences with the reference sequence of the normal human RB1 gene to identify any mutations.

02

(b) Evaluate if osteosarcoma cells produce any pRB protein

1. Extract proteins from the cultured osteosarcoma cells by lysing the cells and performing protein extraction. 2. Perform SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to separate the extracted proteins based on their molecular weights. 3. Perform western blotting on the SDS-PAGE using a primary antibody that specifically binds to the pRB protein. 4. Visualize the presence of the pRB protein with a secondary antibody that reacts with the primary antibody and produces a detectable signal (e.g., chemiluminescence).

03

(c) Evaluate if the addition of a normal RB1 gene changes the cancer-causing potential of osteosarcoma cells

1. Generate a recombinant DNA construct containing the normal human RB1 cDNA under the control of a strong promoter. 2. Introduce the recombinant DNA construct into the cultured osteosarcoma cells using a suitable gene transfer method (e.g., electroporation or viral transduction). 3. Select for stably transfected cells that express the normal RB1 gene. 4. Test the ability of these engineered osteosarcoma cells to form malignant tumors upon injection into the cancer-prone mice. 5. Compare the tumor formation frequency and tumor growth rate between mice injected with engineered osteosarcoma cells (with the normal RB1 gene) and mice injected with non-engineered osteosarcoma cells (without the normal RB1 gene).

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