ABS 116: Role of SV40 in immortalized AALE cells (lung epithelial cells) and its affects on gene expression

Sai Ramani Choudari ¹ , Samyukta Mallick ² , Alison Taylor ²

¹ Barnard College, NY, USA
² Herbert Irving Comprehensive Cancer Center

Van Wickle (2025) Volume 1, ABS 116

Introduction: Increasing developments made in cell culture technology and processes allow for researchers to better understand and replicate cellular development and the basis of many biological systems in-vitro. One key exploration made through new cell culture techniques and technologies is the immortalization of cell lines. Cells can be immortalized via numerous methods such as isolating lines from naturally occurring cancers (i.e. HeLa cells), over-expressing proteins that are important to cell immortality, and introducing viral genes that can disrupt the cell cycle. The method of immortalizing cells via the introduction of the viral gene SV40 has proven to be highly successful, however the implications of immortalizing cells using SV40 viral vectors and its potential effects on gene expression is yet to be fully studied. As such, this research aims to understand the role of SV40 in immortalized AALE (lung epithelial cells) and its impact on gene expression.

Methods: CRISPR/Cas9 was used to knock out SV40 small and large T antigens in AALE cells. The AALE cells are immortalized with SV40, hence, to model a negative control we need to knock out the SV40 small and large T antigens. A fluorescent mCherry tag was also added to allow for easier sorting. This was done to our 3p WT, 3p del, and 3q gain AALE lines. FACS sorting was used to select for those transformed cells that are SV40 KO. ​

Results: Preliminary data from quantitative PCR tests indicated that in SV40+ lines the WNT gene is up-regulated, identifying a key gene pathway for further research. It was also found that SV40 KO leads to decreased cellular proliferation.

Discussion: Currently RNA sequencing is underway to determine the full breadth at which WNT pathways are affected by SV40. This finding paves the way for future cancer research that seeks to uncover how SV40 will interact with cancerous genomic alterations.