Emergence of CRISPR/Cas9 genome editing provides a robust method for gene targeting in a variety of cell types, including fertilized rat embryos. The authors used this method to generate a transgenic rat L1cam knockout model of X-linked hydrocephalus (XLH) with human genetic etiology. The object of this study was to use diffusion tensor imaging (DTI) in studying perivascular white matter tract injury in the rat model and to characterize its pathological definition in histology.
Two guide RNAs designed to disrupt exon 4 of the L1cam gene on the X chromosome were injected into Sprague-Dawley rat embryos. Following embryo transfer into pseudopregnant females, rats were born and their DNA was sequenced for evidence of L1cam mutation. The mutant and control wild-type rats were monitored for growth and hydrocephalus phenotypes. Their macro- and microbrain structures were studied with T2-weighted MRI, DTI, immunohistochemistry, and transmission electron microscopy (TEM).
The authors successfully obtained 2 independent L1cam knockout alleles and 1 missense mutant allele. Hemizygous male mutants from all 3 alleles developed hydrocephalus and delayed development. Significant reductions in fractional anisotropy and axial diffusivity were observed in the corpus callosum, external capsule, and internal capsule at 3 months of age. The mutant rats did not show reactive gliosis by then but exhibited hypomyelination and increased extracellular fluid in the corpus callosum.
The CRISPR/Cas9-mediated genome editing system can be harnessed to efficiently disrupt the L1cam gene in rats for creation of a larger XLH animal model than previously available. This study provides evidence that the early pathology of the periventricular white matter tracts in hydrocephalus can be detected in DTI. Furthermore, TEM-based morphometric analysis of the corpus callosum elucidates the underlying cytopathological changes accompanying hydrocephalus-derived variations in DTI. The CRISPR/Cas9 system offers opportunities to explore novel surgical and imaging techniques on larger mammalian models.
ABBREVIATIONSCas9 = CRISPR-associated endonuclease 9; CRISPR = clustered regularly interspaced short palindromic repeat; CSF = cerebrospinal fluid; Dax = axial diffusivity; Drad = radial diffusivity; DTI = diffusion tensor imaging; FA = fractional anisotropy; GFAP = glial fibrillary acidic protein; gRNA = guide RNA; Iba1 = ionized calcium-binding adapter molecule 1; IHC = immunohistochemistry; KO = knockout; L1CAM = L1 cell adhesion molecule; MD = mean diffusivity; NF-H = neurofilament H; P = postnatal day; ROI = region of interest; SD = standard deviation; SEM = standard error of the mean; TEM = transmission electron microscopy; XLH = X-linked hydrocephalus.
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