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Mice comprising mutations resulting in expression of c-truncated fibrillin-1

A fibril, protein technology, applied in the field of mice containing mutations that cause C-truncated fibrillin-1 expression, can solve the problem that transgenic non-human mammals do not fully reflect the symptoms of NPSCL

Inactive Publication Date: 2019-05-24
REGENERON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently available transgenic non-human mammals engineered to have FBN1 mutations do not adequately reflect the symptoms of NPSCL

Method used

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  • Mice comprising mutations resulting in expression of c-truncated fibrillin-1
  • Mice comprising mutations resulting in expression of c-truncated fibrillin-1
  • Mice comprising mutations resulting in expression of c-truncated fibrillin-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0275] Example 1. Generation of MAID 8501 Fbnl mutant mice with a truncated C-terminus.

[0276] Generation of mutant Fbn1 mouse alleles to reconstitute human mutant Fbn1 alleles. Using NM_007993.2 as the reference sequence, the mutation is c.8213_8214delinsACT. This mutation, created by insertion of A between c.8212 and 8213 and a G>T substitution at c.8214, generates a premature stop codon in the penultimate exon of Fbnl. The mutant allele is denoted MAID 8501. see figure 1 . The mutation is within the last 50 nucleotides of the penultimate exon and is predicted to escape mRNA nonsense-mediated degradation (NMD), thereby causing expression of the mutant truncated fibrillin precursor protein.

[0277] To generate mutant alleles, CRISPR / Cas9 components were introduced into C57BL / 6 one-cell-stage embryos via pronuclear injection or piezoelectric injection of cytoplasmic mRNA along with a donor template. The sequence of the guide RNA DNA targeting sequence is shown in SEQ I...

Embodiment 2

[0281] Example 2. Generation of MAID 8520 Fbn1 mutant mice with a truncated C-terminus.

[0282] In another experiment, guide RNA sequences and donor sequences were designed to generate a mutant Fbn1 allele corresponding to the human c.8155_8156del Fbn1 allele with coding exon 64 (the penultimate exon) A deletion of two base pairs in p.Lys2719 resulted in a frameshift, with a subsequent premature stop codon 17 codons downstream of p.Lys2719. The predicted mutant allele is indicated as MAID 8502. see image 3 .

[0283] To generate mutant alleles, CRISPR / Cas9 components were introduced into C57BL / 6 one-cell-stage embryos via pronuclear injection or piezoelectric injection of cytoplasmic mRNA along with a donor template. One clone produced has image 3 The MAID 8520 mutant allele shown in , rather than the expected MAID 8502 allele. The MAID 8520 mutant allele also causes premature termination of the encoded Fbn1 protein, as image 3 shown in.

[0284] F0 founder mice wer...

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Abstract

Provided are non-human animals comprising a mutation in the Fbnl gene to model neonatal progeroid syndrome with congenital lipodystrophy (NPSCL). Also provided are methods of making such non-human animal models. The non-human animal models can be used for screening compounds for activity in inhibiting or reducing NPSCL or ameliorating NPSCL- like symptoms or screening compounds for activity potentially harmful in promoting or exacerbating NPSCL as well as to provide insights in to the mechanism of NPSCL and potentially new therapeutic and diagnostic targets.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This application claims the benefit of US Application No. 62 / 368,924, filed July 29, 2016, which is hereby incorporated by reference in its entirety for all purposes. [0003] References to Sequence Listings Submitted as Text Files via EFS WEB [0004] The sequence listing written in file 500041SEQLIST.txt is 184 kilobytes, was created on July 28, 2017, and is hereby incorporated by reference. Background technique [0005] More than 3000 mutations have been identified clinically in the human fibrillin-1 (FBN1 ) gene. These mutations have been associated with a variety of conditions, including fibrillinopathy type I, Marfan syndrome, MASS syndrome, simple heterophakia syndrome, thoracic aortic aneurysm, and Weiss-Marfan syndrome Weill-Marches anisyndrome, geleophysic dysplasia and acromicric dysplasia, skin stiffness syndrome, and neonatal progeria-like syndrome with congenital lipodystrophy (NPSCL ). Currently availab...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/027
CPCA01K2217/00A01K67/0275A01K2227/105A01K2267/0306A61K49/0008C07K14/78A01K2217/056
Inventor 查琳·亨特詹森·曼斯塔斯国春·龚卡曼·维纳斯·莱杰斯珀·格罗马达阿里斯·N.·伊科诺米季斯
Owner REGENERON PHARM INC