Abstracts

Rationale: In utero electroporation (IUE) has been widely used to induce specific transgene expression in cerebral cortex. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system has been recently developed to specifically knock out genomic DNA in different species. Here, we are trying to set up a system to combine these two techniques in order to study the functions of PCDH19 in cerebral cortex. Methods: CRISPR PCDH19 plasmids : PX330 vector that express the gRNA and Cas9 in a single expression plasmid was used to design CRISPR PCDH19 targeting the exon 1. The target sites were chosen based on the web-based design program. To examine the knockout efficiency, rat neuroblastoma cells were transfected with PX330 PCDH19 or PX330 control. The genomic knockout efficiency was determined by SURVEYOR assay to (Transgenomic Inc). Briefly, the cells were collected 48 h after transfection and DNA was extracted from the experimental and control group. Equal amount DNA from both groups was mixed for PCR amplification that flanks the targeting sequence. The mixtures of hetero- and homoduplexes were treated with SURVEYOR nuclease that specifically cut both strands of a DNA heteroduplex on the 3'-side of the mismatch site, including insertion/deletion mismatches and all base-substitution mismatches. The DNA fragments were then analyzed by Agarose gel electrophoresis. The formation of new cleavage products, due to the presence of one or more mismatches, will be indicated by the presence of additional bands. In utero electroporation: Pregnant rats were anesthetized at E13 or 14, and uterus were exposed. Lateral ventricles are injected with pulled glass microcapillary needles with plasmids in a 0.01% Fast Green solution using a microinjector. Electrodes were placed on either side of the embryo's head with the positive electrode over the transfection site, and 3×50 ms square pulses at 70 uV are administered at 1 second intervals with a BTX830 square-wave pulse generator (Havard Apparatus). Brains were harvested 5 days after the transfection for migration assay or postnatal 21 days for morphology assay and seizure induction assay. Results: CRISPR PCDH19 plasmids efficiently knocked out PCDH19 in vitro. 2 sgRNA sequences were designed to target genomic PCDH19 exon1. The SURVEYOR primers were designed to amplify a 507 bp DNA fragments spanning the sgRNA targeting sites. CRISPR PCDH19 E1-A generated 341bp and 166bp fragments, and CRISPR PCDH19 E1-B generated 386bp and 121bp fragments. We also showed that 2 days after the in utero electroporation of GFP plasmids, the neurons migrated normally into the subventricular (SVZ) /intermediate zone (IZ). Under higher magnification, these transfected neurons showed appropriate multipolar morphology Conclusions: 1.We designed 2 CRISPR constructs targeting rat PCDH19 thtat are able to efficiently knocked out PCDH19 in vitro. 2.In Utero electroporation can efficiently delivery DNA into rodent embryonic brains. 3.We will combine these two techniques to study the function of PCDH19 in developing brain.