Question

1. You are a new undergraduate researcher in the Ruohola-Baker lab. Your job is work with a postdoc named Shiri. Your job is to test the function of a de novo protein named EED binder (EB). In this aim you acquired DNA for this protein. The construct is cloned into pet28b, a bacterial expression vector. Your goal is to express it in human stem cells. A. What method would you use to extract your DNA fragment from the pet28b plasmid? B. For this experiment Shiri wants you to use a specific viral vector which should integrate into a site designated AAVS1. What viral vector is it and why is it commonly used for gene delivery in-vivo? C. What is the difference between transient and stable transfection? What function did Shiri design EB to have?. EB GFP 22.2-GFP kDa 35 25 GFP

Answer 1

2- Given that EB gene is present in the pET28 vector. So, we need to cut the gene from the pET28b vector and ligate it into some mammalian vector.

To cut the gene we need to digest the vector with the appropriate enzyme such that after digestion we only get the gene upon digestion

2- For cloning, we will use site-specific integration by adeno-associated virus.

The efficiency of stable integration the adeno-associated virus is very high which is up to 90%.

3- In Transient transfection, we can use the transfected cell for maximum 2 generation. It is temporary transfection and we need to transfect the cell if we need to perform another set of experiment.

In Stable transfection, the transfected gene is stably integrated into the genome of the host cell and divide along with the genome of the host cell. We do not need to transfect the cell if we need to perform another set of experiment. It helps us to save time and reagents.

The vector shiri is using will stably integrate the gene in the genome and gives stable

4-

A- Observed molecular weight of the protein is increased by around 15kDa due to GFP tagged

B- EB protein can be probed by using the antibody against the GFP protein.

C- Cells which were transfected by the virus based vector are expressing the EB protein.

5- Upon cells treated with Dox, cells were able to express EB protein and doesn't express when cells were depleted with Dox. This shows that the vector is inducible by Dox.

trimethylation of H3K27 leads to downregulation of nearby genes. When Dox was added in the media, it leads to induction of EB gene. Induction of EB gene expression leads to demethylation of H3K27me3 which can be seen in the lower panel of the western blot where upon dox treatment levels of H3K27me3 is decreased