Research: Humanization of the Promoter Region of FcRn
Awards: WESEF 2023 - "in vitro biology award", Somers Science Fair 2022 -2nd place Medicine and Health
Mentor: Dr. Uccellini and Dr. Mansfield
Research Location: Regeneron
Abstract:
The use of mice to model human immune systems is widespread in pharmaceutical laboratories. This results in the inaccurate representation of human levels of immunoglobulin G (IgG) in mice. Four pRG977 plasmids were constructed in an attempt to create a targeting vector that expresses neonatal crystallizable fragment receptor (FcRn), which is responsible for long IgG half-life in humans and is absent in adult mice, resulting in significantly smaller IgG half-lives. The ubiquitin (Ub) promoter was used as a control promoter in two plasmid and green fluorescence protein (GFP) cDNA was used as a control gene in two plasmids. Therefore, the first plasmid had the Ub promoter and GFP cDNA, second had Ub promoter with FcRn cDNA, third had the FcRn promoter with GFP cDNA and the final plasmid had the FcRn promoter with FcRn cDNA. Plasmids were constructed through restriction enzyme digests and Gibson assembly ligations. Resulting plasmids were transformed by electroporation into E. coli cells, which were screened. Minipreps and maxipreps were performed on the most accurate clones, which were transfected into HEK293T cells. Through flow cytometry, it was determined that 22% of cells expressed FcRn with the ubiquitin promoter and 8% of cells expressed FcRn with the FcRn promoter. Constructing these expression plasmids allows for the development of a more accurate human FcRn expression mouse model.
About this Scientist:
Surbhi Singhal is a junior enrolled in the BioChem magnet at Saunders High School. Her interest in biology was first sparked in eighth grade living environment and she has the privilege of exploring biotechnology and molecular biology through her magnet program and summer internships at Regeneron Pharmaceuticals. In the future, Surbhi hopes to conduct epigenetic research and its applicability in medicine.