Gene Knock Up via 3’UTR editing to study gene functionin vivo

Abstract

Currently available genetic tools do not allow researchers to upregulate ('Knock Up') the levels of a given protein while retaining its cell-type-specific regulation. As a result, we have limited ability to develop overexpression-related disease models, to study the contribution of single genes in diseases caused by copy number variations and to identify disease pathways for drug targets. Here we develop two approaches for endogenous gene upregulation: conditional Knock Up (cKU) utilizing the Cre/lox system, and CRISPR-Cas9 mediated gene Knock Up (KU) in wild-type mouse embryos and human cells. Using glial cell line derived neurotrophic factor (GDNF) as a proof of concept, we show that both approaches resulted in upregulation of endogenous GDNF levels without disturbing Gdnf's expression pattern. Furthermore, CNS-specific GDNF cKU resulted in dopaminergic abnormalities and schizophrenia-like phenotypes. Our results suggest that gene Knock Up can reveal unknown gene functions and provide novel entry points for studying neurological disease.

Increased Endogenous GDNF in Mice Protects Against Age-Related Decline in Neuronal Cholinergic Markers

“…Second, gene expression and correlation analysis were limited to two brain areas i.e., cortex and striatum, because of samples data availability and large size of the dataset required for a reliable multiple correlation analysis. Moreover, given that GDNF is ubiquitously and constitutively expressed in Gdnf wt/hyper mice, future studies should be designed to address the outcome of adult onset increase of endogenous GDNF on cholinergic transmission, including the analysis of conditional GDNF hypermorphic mice ( Mätlik et al, 2019 ).…”