Summary of DNA damage repair is unaffected by mimicked heterozygous levels of BRCA2 in HT-2 cells
This article is about, the human BRCA2 gene, that naturally functions as a tumor suppressor. It is believed that if one or both functional allele(s) of this gene is lost, tumor will be generated consequently. In this article, the scientists tried to prove that the above mentioned statement is true. So, they mimicked this human BRCA2 gene and result was quite amazing. They saw that DNA damage and repair system was not affected by this mimicked BRCA2 gene. Also they found out that, the level of p53 (a tumor suppressor protein) and p27 (cell cycle inhibitor protein) was increased when the BRCA2 function is lost.
The human BRCA2 gene encodes a nuclear protein of 3,418 amino acids, and is believed to play a pivotal role in DNA damage repair. In this experiment, BRCA2 protein has been shown to bind to RAD51, the mammalian homologue of the RecA recombinase, and thus is believed to be involved in the repair of DNA double-strand breaks. Moreover, BRCA2 regulates both the DNA binding ability of RAD51 and its intracellular location. The scientists have utilized HT-29 colon cancer cells in this experiment and they mimicked the heterozygous state of BRCA2 in these cells through RNA interference.
Materials and methods:
1. Retroviral vectors:
They utilized pRETRO-SUPER plasmid for short hairpin RNA targeting of BRCA2 and confirmed all plasmids by DNA sequencing and/or restriction digestion.
2. Cell culture and retro viral infection:
Firstly, they grew HT-29 cells in Dulbecco’s modification of Eagle’s medium. Produced supernatant and then incubated them overnight. After that, infected them with empty vector, control retrovirus (control RNAi cells). After 48 hours they passed infected HT-29 cells into selection media and continued selection process for 14 days.
3. Western blotting: