Question

Two stable cell lines generated from the RAS oncogene are cancerous, and now, with the ubiquitin thiolesterase (UCHL1) gene over-expression.

1. Propose a hypothesis to explain the differences in transient vs stable overexpression of the UCHL1 gene results.

2. Next, propose a series of experiments to test your hypothesis with controls.

Answer 1

1. Transiently transfected cells with UCHL1 gene expresses the UCHL1 but don't integrate with the genome.In this set up,UCHL1 gene will not be replicated.The expression of the UCHL1 will be lost after cell divison.But,RAS oncogene experssion will be maintained because it is integrated into the genome. As a result,it promotes the upregulation of AKT and further ERK1/2 protein synthesis.

Stable overexpression of the RAS and UCHL1 gene togeher can integrate into genome and replicate further.Stable coexpresssion of the RAS and UCHL1 can occur simultaneously.As a result of these two proteins co-expression,it stably increase the ERK1/2 abundantly compare to the transient UCHL1 expression.

2. Experiments suggested to test the hypothesis

Control 1 -Ras overexpression with no UCHL1 transfection

Control 2- UCHL1 overexpression with no Ras tranfection

test - Ras and UCHL1 coexpression- stable and transient

Western blot analysis experiment -Protein blots for the ERK1/2,Ras and UCHL1 protein and internal control GAPDH.