You have been tasked with creating a transgenic human cell line that under-expre

Question

You have been tasked with creating a transgenic human cell line that under-expresses Rac1, a G-protein with many roles in signal transduction. Previous research has shown that a precise series of 5 nucleotide changes in a 50 bp stretch upstream of the Rac1 gene modifies an enhancer region and reduces transcription rate of Rac1. This research was done in HeLa cells, but you wish to modify HEK 293T (kidney-derived) cells.

Explain your strategy for creating the transformed HEK 293T with the above modification. What will you need to find out about the genomic DNA sequence of the cells? What existing tools can you use, and what types of molecules will you need to custom synthesize? How will you confirm a successful, functional transformation? In 2-3 paragraphs, please lay out the steps that you would take, addressing the questions above.

Ans. I am planning to use Crispr/cas9 system, by transfroming the cell line expression from Hela to HEK 293T, but I am not sure if that is the right idea? plz help

Explanation / Answer

To produce mutation in lab, site directed mutagenesis is used. In site directed mutagenesis, mjtation can be created intentionally. To change the 5 nucleotide in the 50bp stretch upstream of Rac1 gene, CRISPER / cas9 system can be utilized in HEK 293T cell. CIRSPER/ cas9 method consists of two componant, A endonuclease known as CRISPER associated endonuclease and another guide RNA.

Here, the guide RNA will be short sequece of Rac1 gene, where the 5 nucleoide will be changed in the 50bp sequence. HEK 293 T cell will be transfected with gRNA with the modified Rac1 sequence. Inside the cell gRNA will bind where it will find the target Rac1 gene. The endonuclease will cut the genomic DNA and the transfeted Rac1 sequence will hybridize to the rac1 gene inside the HEK 293 cells

To confirm whether CRISPER/ cas9 has been able to introduce the mutaiton, we can perform RT-PCR for the expression analysis of Rac1 gene. When the enhancer region is modified it will reduce the transcription rate of Rac1. In normal HEK 293T cell, Rac1 will be highly expressed in comparison to mutated HEK293T cell which is produce less amount of Rac1 mRNA.

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