Lysophosphatidic acid (LPA) is a lipid growth factor that exerts diverse biological effects through its cognate receptors (LPA 1 -LPA 6 ). LPA 1 , which is predominantly expressed in the brain, plays a pivotal role in brain development. However, the role of LPA 1 in neuronal migration has not yet been fully elucidated. Here, we delivered LPA 1 to mouse cerebral cortex using in utero electroporation. We demonstrated that neuronal migration in the cerebral cortex was not affected by the overexpression of LPA 1 . Moreover, these results can be applied to the identification of the localization of LPA 1 . The subcellular localization of LPA 1 was endogenously present in the perinuclear area, and overexpressed LPA 1 was located in the plasma membrane. Furthermore, LPA 1 in developing mouse cerebral cortex was mainly expressed in the ventricular zone and the cortical plate. In summary, the overexpression of LPA 1 did not affect neuronal migration, and the protein expression of LPA 1 was mainly located in the ventricular zone and cortical plate within the developing mouse cerebral cortex. These studies have provided information on the role of LPA 1 in brain development and on the technical advantages of in utero electroporation.
Corneal dystrophy is an autosomal dominant disorder caused by mutations of the transforming growth factor β-induced ( TGFBI ) gene on chromosome 5q31.8. This disease is therefore ideally suited for gene therapy using genome-editing technology. Here, we isolated human limbal epithelial stem cells (ABCG2+/ABCB5+ double-positive LESCs) and established a TGFBI knockout using RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing. An LESC clone generated with a single-guide RNA (sgRNA) targeting exon 4 of the TGFBI gene was sequenced in order to identify potential genomic insertions and deletions near the Cas9/sgRNA-target sites. A detailed analysis of the differences between wild type LESCs and the single LESC clone modified by the TGFBI -targeting sgRNA revealed two distinct mutations, an 8 bp deletion and a 14 bp deletion flanked by a single point mutation. These mutations each lead to a frameshift missense mutation and generate premature stop codons downstream in exon 4. To validate the TGFBI knockout LESC clone, we used single cell culture to isolate four individual sub-clones, each of which was found to possess both mutations present in the parent clone, indicating that the population is homogenous. Furthermore, we confirmed that TGFBI protein expression is abolished in the TGFBI knockout LESC clone using western blot analysis. Collectively, our results suggest that genome editing of TGFBI in LESCs by CRISPR/Cas9 may be useful strategy to treat corneal dystrophy.
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