Constructing Plasmids for “Fluorescent Mice”

Overview

The purpose of this project is to insert H2B into pBlueScript/IRES-GFP/BFP to construct pBlueScript/IRES-H2BGFP/BFP plasmids. GFP/BFP stands for green/blue fluorescence protein, and could be directly visualized when exposed to light in the blue to UV range. H2B (Histone 2B) functions in the nucleus and possesses signals that allow nucleus localization. Fusing it with GFP/BFP creates a brighter image because the fluorescence would be concentrated in the nucleus as opposed to dispersed in the cytoplasm. The constructed plasmids will be used to insert IRES-H2BGFP/BFP into an endogenous locus of gene of interest, which will allow researchers to track and visualize gene expression in live cells or even in live mice through examining green or blue fluorescence . The H2B fragment was obtained through PCR. Then, Restriction Digestion was performed using Pci1 and Nco1 to cut the H2B fragments and the pBlueScript/IRES-GFP/BFP respectively. The digested fragments were ligated together and transformed into Ecoli for plasmid amplification. Plasmids were purified and sent out for sequencing. Sanger Sequencing confirmed that the H2B inserts were correct without any mutations. In conclusion, H2B was successfully inserted into the pBlueScript/IRES-GFP/BFP plasmids. Two new plasmids, pBlueScript/IRES-H2BGFP and pBlueScript/IRES-H2BBFP, were constructed and will be used to track gene expression in “fluorescent mice”.

Competition history

• AJAS 2018 Category not listed

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