Team:NUS Singapore-Sci/Reporter GLB
Reporter System
GLB
GLB1 encodes a human lysosomal acid β-galactosidase (GLB), an enzyme that is responsible for the cleavage of terminal β-linked galactose residues from glycoproteins, sphingolipids, keratan sulfate, and other glycoconjugates. The loss of GLB in physiological systems will result in autosomal recessive lysosomal storage diseases such as GM1 gangliosidosis and Morquio B disease (Suzuki et al., 2001).
Over hundreds of single nucleotide mutations have been implicated in this disease. One of the challenges in building a disease model lies in manipulating GLB1 without affecting normal cellular function since it is an endogenous housekeeping gene. The GLB1 deficient fibroblasts may not be easily available for experimental work. Moreover, while the E. coli β-galactosidase gene LacZ is widely used as a reporter in mammalian systems, this bacterial ortholog cannot be reliably used to create accurate representations of human GLB diseases caused by mutations in the gene. Therefore, we want to investigate if overexpression of exogenous GLB1 is possible in mammalian cell lines. We hope to use such an overexpression system as a disease model to test the usefulness of our RESCUE editor, Cas13b-APOBEC to restore the functions of GLB. Furthermore, we aimed to generate a C to T point mutation in GLB1 that causes a loss of enzymatic function without affecting its translation and degradation.
Over hundreds of single nucleotide mutations have been implicated in this disease. One of the challenges in building a disease model lies in manipulating GLB1 without affecting normal cellular function since it is an endogenous housekeeping gene. The GLB1 deficient fibroblasts may not be easily available for experimental work. Moreover, while the E. coli β-galactosidase gene LacZ is widely used as a reporter in mammalian systems, this bacterial ortholog cannot be reliably used to create accurate representations of human GLB diseases caused by mutations in the gene. Therefore, we want to investigate if overexpression of exogenous GLB1 is possible in mammalian cell lines. We hope to use such an overexpression system as a disease model to test the usefulness of our RESCUE editor, Cas13b-APOBEC to restore the functions of GLB. Furthermore, we aimed to generate a C to T point mutation in GLB1 that causes a loss of enzymatic function without affecting its translation and degradation.