Bacteriophage gene targeting vectors generated by transplacement

A rate-determining step in gene targeting is the generation of the targeting vector. We have developed bacteriophage gene targeting vectorology, which shortens the timeline of targeting vector construction. Using retro-recombination screening, we can rapidly isolate targeting vectors from an embryonic stem cell genomic library via integrative and excisive recombination. We have demonstrated that recombination can be used to introduce specific point mutations or unique restriction sites into gene targeting vectors via transplacement. Using the choline/ethanolamine kinase alpha and beta genes as models, we demonstrate that transplacement can also be used to introduce specifically a neo resistance cassette into a gene targeting phage. In our experience, the lambdaTK gene targeting system offers considerable flexibility and efficiency in TV construction, which makes generating multiple vectors in one week's time possible.     

Differences between immunodeficient mice generated by classical gene targeting and CRISPR/Cas9-mediated gene knockout

Immunodeficient mice are widely used for pre-clinical studies to understand various human diseases. Here, we report the generation of four immunodeficient mouse models using CRISPR/Cas9 system without inserting any foreign gene sequences such as Neo^R cassettes and their characterization. By eliminating any possible effects of adding a Neo^R cassette, our mouse models may allow us to better elucidate the in vivo functions of each gene. Our FVB-Rag2^?/?, B6-Rag2^?/?, and BALB/c-Prkdc^?/? mice showed phenotypes similar to those of the earlier immunodeficient mouse models, including a lack of mature B cells and T cells and an increase in the number of CD45⁺DX-5⁺ natural killer cells. However, B6-Il2rg^?/? mice had a unique phenotype, with a lack of mature B cells, increased number of T cells, and decreased number of natural killer cells. Additionally, serum immunoglobulin levels in all four immunodeficient mouse models were significantly reduced when compared to those in wild-type mice with the exception of IgM in B6-Il2rg^?/? mice. These results indicate that our immunodeficient mouse models are a robust tool for in vivo studies of the immune system and will provide new insights into the variation in phenotypic outcomes resulting from different gene-targeting methodologies.     

Identification and functional consequences of a new mutation (E155G) in the gene for GCAP1 that causes autosomal dominant cone dystrophy

Mutations in the gene for guanylate cyclase-activating protein-1 (GCAP1) (GUCA1A) have been associated with autosomal dominant cone dystrophy (COD3). In the present study, a severe disease phenotype in a large white family was initially shown to map to chromosome 6p21.1, the location of GUCA1A. Subsequent single-stranded conformation polymorphism analysis and direct sequencing revealed an A464G transition, causing an E155G substitution within the EF4 domain of GCAP1. Modeling of the protein structure shows that the mutation eliminates a bidentate amino acid side chain essential for Ca2+ binding. This represents the first disease-associated mutation in GCAP1, or any neuron-specific calcium-binding protein within an EF-hand domain, that directly coordinates Ca2+. The functional consequences of this substitution were investigated in an in vitro assay of retinal guanylate cyclase activation. The mutant protein activates the cyclase at low Ca2+ concentrations but fails to inactivate at high Ca2+ concentrations. The overall effect of this would be the constitutive activation of guanylate cyclase in photoreceptors, even at the high Ca2+ concentrations of the dark-adapted state, which may explain the dominant disease phenotype.     

Efficient gene targeting in mouse zygotes mediated by CRISPR/Cas9-protein

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9–FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.     

Correction of glycogen storage disease type 1a in a mouse model by gene therapy

Glycogen storage disease type 1a (GSD-1a), characterized by hypoglycemia, liver and kidney enlargement, growth retardation, hyperlipidemia, and hyperuricemia, is caused by a deficiency in glucose-6-phosphatase (G6Pase), a key enzyme in glucose homeostasis. To evaluate the feasibility of gene replacement therapy for GSD-1a, we have infused adenoviral vector containing the murine G6Pase gene (Ad-mG6Pase) into G6Pase-deficient (G6Pase(-/-)) mice that manifest symptoms characteristic of human GSD-1a. Whereas <15% of G6Pase(-/-) mice under glucose therapy survived weaning, a 100% survival rate was achieved when G6Pase(-/-) mice were infused with Ad-mG6Pase, 90% of which lived to 3 months of age. Hepatic G6Pase activity in Ad-mG6Pase-infused mice was restored to 19% of that in G6Pase(+/+) mice at 7-14 days post-infusion; the activity persisted for at least 70 days. Ad-mG6Pase infusion also greatly improved growth of G6Pase(-/-) mice and normalized plasma glucose, cholesterol, triglyceride, and uric acid profiles. Furthermore, liver and kidney enlargement was less pronounced with near-normal levels of glycogen depositions in both organs. Our data demonstrate that a single administration of a recombinant adenoviral vector can alleviate the pathological manifestations of GSD-1a in mice, suggesting that this disorder in humans can potentially be corrected by gene therapy.     

X-linked cone-rod dystrophy (locus COD1): identification of mutations in RPGR exon ORF15

X-linked cone-rod dystrophy (COD1) is a retinal disease that primarily affects the cone photoreceptors; the disease was originally mapped to a limited region of Xp11.4. We evaluated the three families from our original study with new markers and clinically reassessed all key recombinants; we determined that the critical intervals in families 2 and 3 overlapped the RP3 locus and that a status change (from affected to probably unaffected) of a key recombinant individual in family 1 also reassigned the disease locus to include RP3 as well. Mutation analysis of the entire RPGR coding region identified two different 2-nucleotide (nt) deletions in ORF15, in family 2 (delAG) and in families 1 and 3 (delGG), both of which result in a frameshift leading to altered amino acid structure and early termination. In addition, an independent individual with X-linked cone-rod dystrophy demonstrated a 1-nt insertion (insA) in ORF15. The presence of three distinct mutations associated with the same disease phenotype provides strong evidence that mutations in RPGR exon ORF15 are responsible for COD1. Genetic heterogeneity was observed in three other families, including the identification of an in-frame 12-nt deletion polymorphism in ORF15 that did not segregate with the disease in one of these families.     

Mutations in the ABCA4 (ABCR) gene are the major cause of autosomal recessive cone-rod dystrophy

The photoreceptor cell-specific ATP-binding cassette transporter gene (ABCA4; previously denoted "ABCR") is mutated, in most patients, with autosomal recessive (AR) Stargardt disease (STGD1) or fundus flavimaculatus (FFM). In addition, a few cases with AR retinitis pigmentosa (RP) and AR cone-rod dystrophy (CRD) have been found to have ABCA4 mutations. To evaluate the importance of the ABCA4 gene as a cause of AR CRD, we selected 5 patients with AR CRD and 15 patients from Germany and The Netherlands with isolated CRD. Single-strand conformation-polymorphism analysis and sequencing revealed 19 ABCA4 mutations in 13 (65%) of 20 patients. In six patients, mutations were identified in both ABCA4 alleles; in seven patients, mutations were detected in one allele. One complex ABCA4 allele (L541P;A1038V) was found exclusively in German patients with CRD; one patient carried this complex allele homozygously, and five others were compound heterozygous. These findings suggest that mutations in the ABCA4 gene are the major cause of AR CRD. A primary role of the ABCA4 gene in STGD1/FFM and AR CRD, together with the gene's involvement in an as-yet-unknown proportion of cases with AR RP, strengthens the idea that mutations in the ABCA4 gene could be the most frequent cause of inherited retinal dystrophy in humans.     

Regulation of gene expression by the CYP27B1 promoter-study of a transgenic mouse model

The enzyme 25-hydroxyvitamin D 1-hydroxylase (CYP27B1) is the rate limiting enzyme in the two-step activation process of Vitamin D to its active form 1,25-dihydroxyvitamin D (1,25D) and is located in the mitochondrial fraction of the proximal tubular cells of the kidney. More recently CYP27B1 activity and expression have also been identified in a number of non-renal cells, which is suggestive of new, previously unidentified roles for Vitamin D in the human body. Although the regulation of CYP27B1 activity and expression has been a major focus of interest over the past decades, the exact molecular mechanism behind the regulation of CYP27B1 activity and expression and the role of the CYP27B1 promoter, herein, are still poorly understood. In this study, we created a transgenic mouse model that expresses the luciferase reporter gene under the control of the full-length, 1.5kb, human CYP27B1 promoter. This animal model allows us to study in vivo the tissue-specific, CYP27B1 promoter-controlled, regulation of the expression of the CYP27B1 gene.     

Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells

We mutated, by gene targeting, the endogenous hypoxanthine phosphoribosyl transferase (HPRT) gene in mouse embryo-derived stem (ES) cells. A specialized construct of the neomycin resistance (neor) gene was introduced into an exon of a cloned fragment of the Hprt gene and used to transfect ES cells. Among the G418r colonies, 1/1000 were also resistant to the base analog 6-thioguanine (6-TG). The G418r, 6-TGr cells were all shown to be Hprt- as the result of homologous recombination with the exogenous, neor-containing, Hprt sequences. We have compared the gene-targeting efficiencies of two classes of neor-Hprt recombinant vectors: those that replace the endogenous sequence with the exogenous sequence and those that insert the exogenous sequence into the endogenous sequence. The targeting efficiencies of both classes of vectors are strongly dependent upon the extent of homology between exogenous and endogenous sequences. The protocol described herein should be useful for targeting mutations into any gene.     

Canine RPGRIP1 mutation establishes cone-rod dystrophy in miniature longhaired dachshunds as a homologue of human Leber congenital amaurosis

Cone-rod dystrophy 1 (cord1) is a recessive condition that occurs naturally in miniature longhaired dachshunds (MLHDs). We mapped the cord1 locus to a region of canine chromosome CFA15 that is syntenic with a region of human chromosome 14 (HSA14q11.2) containing the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) gene. Mutations in RPGRIP1 have been shown to cause Leber congenital amaurosis, a group of retinal dystrophies that represent the most common genetic causes of congenital visual impairment in infants and children. Using the newly available canine genome sequence we sequenced RPGRIP1 in affected and carrier MLHDs and identified a 44-nucleotide insertion in exon 2 that alters the reading frame and introduces a premature stop codon. All affected and carrier dogs within an extended inbred pedigree were homozygous and heterozygous, respectively, for the mutation. We conclude the mutation is responsible for cord1 and demonstrate that this canine disease is a valuable model for exploring disease mechanisms and potential therapies for human Leber congenital amaurosis.