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YPIE Scientist: Surbhi Singhal




Research: The Humanization of the Promoter Region of the Neonatal Fc Receptor


Mentor: Scott Mansfield and Melissa Uccellini


Awards: Society for In Vitro Biology Award (WESEF 2023) Carl Zeiss Microscopy Award, Fourth Place in Medicine and Health (WESEF 2024)


Research Location: Regeneron Pharmaceuticals


Abstract:

The use of mice to model human immune systems is widespread in pharmaceutical laboratories because they are cost-effective. However, there are still differences between human and mouse gene expression patterns. This results in limitations, including the inaccurate representation of human levels of immunoglobulin G (IgG) in mice. Neonatal Fc receptor (FcRn) is responsible for long IgG half-life in humans and is absent in adult mice, resulting in smaller IgG half-lives. A targeting vector that expresses FcRn was created to solve this problem. Four pRG977 plasmids were constructed to verify the effectiveness of the human neonatal Fc receptor (hFcRn) promoter in expressing FcRn cDNA. The ubiquitin (Ub) promoter was used as a control promoter in two plasmids and green fluorescence protein (GFP) cDNA was used as a control gene in two plasmids. Therefore, the four plasmids in this study were: (1) Ub promoter and GFP cDNA, (2) Ub promoter and FcRn cDNA, (3) FcRn promoter and GFP cDNA, and (4) FcRn promoter and 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 through PCR. Mini-preps and maxi-preps of the most accurate clones were transfected into HEK293T cells. Through flow cytometry, it was determined that 22% of cells expressed FcRn with the Ub promoter and 8% of cells expressed FcRn with the FcRn promoter. Constructing these expression plasmids allows for verification of the functionality of the hFcRn promoter. The hFcRn promoter and human FcRn gene (FCGRT) exons 1 and 2 were inserted into a bacterial artificial chromosome (bac) containing mouse homology boxes and FCGRT exons 3, 4, and 5.



About this Scientist:

Surbhi Singhal is a senior in the bio-chem technology program at Saunders High School. Outside of science research she is involved in Asian Club and has trained as a Kathak dancer for seven years. Surbhi plans on majoring in Biomedical Engineering in college

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