Genotyping of CRISPR-Edited Mice and Axonal Localization Patterns of Calmodulin 1 3’UTR Isoforms
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Calmodulin (CaM) is a key calcium-signal transducer in multiple cell types and is known to influence neuron differentiation, development, and maturation. Calmodulin 1 (Calm1) is one of many genes that encode for the CaM protein. Because this gene is subject to alternative polyadenylation (APA), a common mechanism for increasing transcript diversity, it produces transcripts with varying lengths of 3' Untranslated Regions (3'UTRs). In Calm1, APA generates isoforms of either long 3'UTR or short 3'UTR. While it is known that the long 3'UTR isoform (Calm1-L) is expressed in neural tissues, the function of Calm1-L is still unknown. To study the function of Calm1-L, the genome editing system CRISPR/Cas9 was utilized to create a mouse model with a deletion of the extended region of the long isoform in mice. Through genotyping PCR, we confirmed that the biogenesis of Calm1-L was impaired. Interbreeding of CRISPR/Cas9 knockout mice produced three genotypes Calm1 WT ?3'UTR, homozygous ?3'UTR, and heterozygous ?3'UTR. The long 3'UTR region has been known to provide an additional regulatory mechanism for mediation axonal and dendritic localization of mRNA transcripts. To test if this mechanism also applies to Calm1, we utilized RNAscope fluorescent in situ hybridization (FISH) with probes designed for the Calm1-L 3'UTR region. Through the application of FISH on WT mice hippocampal cells, we are able to show the differential transcript localization of Calm1-L versus Calm1-S.