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Characterization of a Novel Rat Model of X-linked

Hydrocephalus by CRISPR-mediated mutation in L1cam

A. Scott Emmert1, Shawn M. Vuong1, Crystal Shula1, Diana Lindquist3, Weihong Yuan3, Yueh-Chiang Hu2, Francesco T. Mangano1, and June Goto1
1Division of Pediatric Neurosurgery, 2Division of Developmental Biology, and 3Division of Radiology, Cincinnati Children’s Hospital Medical Center

Overview Generation of L1cam knockout rats Tracking progression of hydrocephalus with Identification of histopathological
Introduction using CRISPR/Cas9 genome editing advanced MR imaging changes in the corpus callosum
• Emergence of the CRISPR/Cas9 genome editing technology
provides a robust method for gene targeting in a variety of cell
types, including fertilized rat embryos.
• We used this method to generate a transgenic rat model of juvenile-
onset hydrocephalus.
• The object of this study was to use the CRISPR/Cas9 system to
knockout the L1cam gene to create a rat model of X-linked
hydrocephalus (XLH).
• We successfully obtained two independent L1cam knockout alleles
and one missense mutant allele.
• Hemizygous male mutants from all three 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. A, gRNAs generated to selectively disrupt the L1 cell adhesion molecule (L1cam) gene in rat
embryos. B, DNA Sanger sequencing traces of the three different mutations identified in the
Conclusions offspring following the CRISPR/Cas9 genome editing. The three characteristic mutations are a
• The CRISPR/Cas9-mediated genome editing system can be single thymine (T) insertion resulting in a premature stop codon (L1camc.206_207insT/y), a large
genomic deletion resulting in a premature stop codon (L1camc.205_505del/y), and a dual amino
harnessed to efficiently disrupt the L1cam gene in rats for creation acid substitution (L1camc.207_209delGACinsTGT/y) changing a tryptophan (W) and threonine (T) to a
of a larger XLH animal model than previously available. cysteine (C) and valine (V), respectively. C, Immunoblot analysis of L1 cell adhesion molecule
(L1CAM) protein expression (~200 kDa and ~70 kDa) in the brains of L1cam knockout
• This study provides evidence that the early pathology of the (KO) rats at ages postnatal day 19 (P19), P21, and P134. Arrows, L1cam protein; asterisk,
periventricular white matter tracts in juvenile-onset hydrocephalus possible cross-reaction of the L1CAM antibody with Ng-cam.
can be detected in DTI. Representative multichannel and single-channel photomicrographs of immunofluorescently marked
astrocytes expressing GFAP (B and E), microglia expressing Iba1 (C and F) (40x), and axons expressing
• Furthermore, TEM-based morphometric analysis of the corpus SMI-32 (L and O) and NF-H (M and P) (60x crop) in the corpus callosum of wild-type (A-C, K-
callosum sheds new light on the underlying cytopathological Depletion of L1CAM protein in M) and L1cam KO (D-F, N-P) rats. Quantification of cell signal intensity of GFAP and Iba1 expressed by
glial cells (G) and SMI-32 and NF-H expressed in axons within the corpus callosum (J). *P<.05,
changes accompanying hydrocephalus-derived variations in DTI. periventricular white matter structures A, Representative ROI set drawn on the corpus callosum (CC), right external capsule (REC), left external ns=nonsignificant, student’s t-test. Numbers are mean ± SEM.
capsule (LEC), left internal capsule (LIC), and right internal capsule (RIC) in a coronal, fractional anisotropy-
• The CRISPR/Cas9 system offers opportunities to explore novel based color-coded axis map of DTI in a control rat at age P97. B, Coronal T2-weighted MRI of the fourth
surgical and imaging techniques on larger mammalian models. ventricle (a-d) and lateral ventricles (e-h) in wild-type (a-b, e-f) and L1cam KO (c-d, g-h) rats at ages postnatal
day 21 (P21) and postnatal day 97 (P97). Area analysis of the fourth ventricle (C) and lateral ventricles (D)
in wild-type and L1cam KO rats as a function of age from P13 through P97.


• 2 guide RNAs, designed to disrupt exon 4 of L1cam on the X

chromosome, were injected into Sprague Dawley rat embryos.
• Following embryo transfer into pseudopregnant females, rats were
born and sequenced for evidence of L1cam mutation.
• Mutant and control rats were monitored for their growth and the Representative photomicrographs (x2, a-f) of 3,3’-diaminobenzidine (DAB) staining of the
corpus callosum (a, d, g, j), stria medullaris and fimbria (a-c), internal capsule (b-c, e, h-k), Radar chart representation of fractional anisotropy (FA), axial diffusivity (Dax), radial diffusivity (Drad), and
XLH phenotype. mean diffusivity (MD) extrapolated from the corpus callosum (A and D), external capsule (B and E), and A, Number of myelinated and unmyelinated axons per TEM field of the corpus callosum in wild-type and
and globus pallidus (b-c, e-f, i-l) stained for L1CAM protein expression in the brains of wild-
• Their macro- and micro-brain structures were studied with T2 MRI, type (a-c, g-i) and L1cam KO rats (d-f, j-l). Some areas are shown at higher magnification internal capsule (C and F) of L1cam KO and wild-type rats at ages of P96-P98 (A-C) and P59-P61 (D-F). The L1cam KO rats. B, Representative electron micrographs and g-ratios of corpus callosum axons in wild-
percent difference between mean DTI measurements from L1cam KO and wild-type rats is presented for type and L1cam KO rats. C, Representative electron micrographs and area measurements of extracellular
diffusion tensor imaging (DTI), immunohistochemistry, and (20x: g-l). Lack of brown staining in the brains of L1cam KO rats indicates absence of L1CAM
each DTI parameter that indicated a significant change upon statistical analysis. *P<.05, **P<.01, fluid (silhouettes) between axons in the corpus callosum of wild-type and L1cam KO rats. *P<.05,
in this model.
transmission electron microscopy (TEM). ns=nonsignificant, student’s t-test. ns=nonsignificant, student’s t-test. Numbers are mean ± SEM.