Talk:20.109(F09): Mod 1 Day 7 Lipofection

Things to Consider

Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to death

READ THE ABSTRACT

The in class discussion will focus on Figures 1, 2, 3, and 6. You should read the whole paper, but focus on the following:

Read the section Strategy of Rad51 disruption carefully to understand Fig 1, and the rest of that page to understand Fig 2.

Be sure to read the section Deletion of Rad51 results in an accumulation of cells in the G2/M phase and subsequent cell death to be able to understand figure 3.

Be sure to read the second paragraph of the Rad51-deficient cells display highly elevated frequencies of chromosomal breakage to get at Fig 6.

Be sure to read Chromosomal breaks may occur during DNA replication in RAD51 deficient cells.

Don't focus on materials and methods section.

Some questions to think about:

• Why and how were cells stuck in G2/M phase?
• How does phase of cell cycle correlate to amount of DNA in cell?

• Is this system tet-on or tet-off activated?
• Why did they do a co-transfection on step 2 of Fig 1? (purpose of each construct)
• How did they ensure that they knocked out both of the Rad51-/- alleles? What method did they use to visualize it?

• What was the targeting and selection strategy of clones (positive and negative selection)?
• How did the group confirm that their clones were properly targeted?

• Colcemid inhibits microtubule formation. What part of the cell cycle does the cell get stuck in if exposed to colcemid?
• What happens to the chromosomes?

• Rather than repairing damage caused by radiation, the Rad-/- cells have DNA that looks worse as time goes on. (fig 6) Why do you think this is?