Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models

Devoy, Anny; Price, Georgia; Giorgio, Francesca De; Bunton-Stasyshyn, Rosie; Thompson, David B.; Gasco, Samanta; Allan, Alasdair; Codner, Gemma; Nair, R.; Tibbit, Charlotte; McLeod, Ross; Ali, Zeinab; Noda, Judith; Marrero‐Gagliardi, Alessandro; Brito-Armas, José M.; Öztürk, Muhammet M.; Simon, Michelle; O’Neill, Edward T.; Bryce-Smith, Sam; Harrison, Jackie; Atkins, Gemma; Corrochano, Silvia; Stewart, Michelle; Teboul, Lydia; Acevedo-Arozena, Abraham; Fisher, Elizabeth; Cunningham, Thomas J.

doi:10.1016/j.isci.2021.103463

Cited by 7 publications

(7 citation statements)

References 69 publications

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“…We partially humanized the Col6a1 gene as a knock-in allele (termed "h"). Like previously generated humanized mouse models, where the canonical splice sites within a humanized gene were faithfully recognized by the mouse splicing machinery (14)(15)(16), the h allele in our model expressed full-length and functional chimeric mouse/human Col6a1 transcripts. Modeling intronic and splice-altering variants, such as the c.930+189C>T, in humanized backgrounds can be more challenging than only inserting a humanized sequence, however (19,20).…”

Section: Discussionmentioning

confidence: 99%

“…While the process for obtaining these humanized loci was previously tedious, with the currently available genome editing technologies it can be achieved rapidly (12, 13). Entire – or nearly entire – human genes have thus been successfully introduced, and expressed, either in a transgenic location (14), or at the endogenous locus in the trans-species genome, substituting their orthologous counterpart (15). Partial genomic humanization has also been successfully applied (16).…”

Section: Introductionmentioning

confidence: 99%

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A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

Bolduc,

Guirguis,

Lubben

et al. 2024

Preprint

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Antisense therapeutics such as splice-modulating antisense oligonucleotides (ASOs) are promising tools to treat diseases caused by splice-altering intronic variants. However, their testing in animal models is hampered by the generally poor sequence conservation of the intervening sequences between human and other species. Here we aimed to model in the mouse a recurrent, deep-intronic, splice-activating,COL6A1variant, associated with a severe form of Collagen VI-related muscular dystrophies (COL6-RDs), for the purpose of testing human-ready antisense therapeuticsin vivo. The variant, c.930+189C>T, creates a donor splice site and inserts a 72-nt-long pseudoexon, which, when translated, acts in a dominant-negative manner, but which can be skipped with ASOs. We created a unique humanized mouse allele (designated as ″h″), in which a 1.9 kb of the mouse genomic region encoding the amino-terminus (N-) of the triple helical (TH) domain of collagen α1(VI) was swapped for the human orthologous sequence. In addition, we also created an allele that carries the c.930+189C>T variant on the same humanized knock-in sequence (designated as ″h+189T″). We show that in both models, the human exons are spliced seamlessly with the mouse exons to generate a chimeric mouse-human collagen α1(VI) protein. In homozygousCol6a1h+189T/ h+189Tmice, the pseudoexon is expressed at levels comparable to those observed in heterozygous patients′ muscle biopsies. WhileCol6a1h/hmice do not show any phenotype compared to wild-type animals,Col6a1h/ h+189TandCol6a1h+189T/ h+189Tmice have smaller muscle masses and display grip strength deficits detectable as early as 4 weeks of age. The pathogenic h+189T humanized knock-in mouse allele thus recapitulates the pathogenic splicing defects seen in patients′ biopsies and allows testing of human-ready precision antisense therapeutics aimed at skipping the pseudoexon. Given that theCOL6A1N-TH region is a hot-spot for COL6-RD variants, the humanized knock-in mouse model can be utilized as a template to introduce otherCOL6A1pathogenic variants. This unique humanized mouse model thus represents a valuable tool for the development of antisense therapeutics for COL6-RD.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

Bolduc,

Guirguis,

Lubben

et al. 2024

Preprint

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“…Samplix sequencing was performed as previously described ( Devoy et al, 2021 ) using primers specific to this project ( Table S3 ). High molecular mass genomic DNA from prospective founder mice was first encapsulated into picolitre-sized droplets using an Xdrop microfluidics device (Samplix).…”

Section: Methodsmentioning

confidence: 99%

Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders

Crombie,

Korecki,

Cleverley

et al. 2024

Disease Models &Amp; Mechanisms

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The TATA-box binding protein-associated factor 1 (TAF1) is a ubiquitously expressed protein and the largest subunit of basal transcription factor TFIID, which plays a key role in initiation of RNA polymerase II-dependent transcription. TAF1 missense variants in males cause X-linked intellectual disability, a neurodevelopmental disorder, and TAF1 is dysregulated in X-linked Dystonia–Parkinsonism, a neurodegenerative disorder. However, this field has suffered from the lack of a genetic mouse model of TAF1 disease to explore mammalian mechanism and treatments. Here, we generated and validated a conditional cre-lox allele, and the first ubiquitous Taf1 knock-out mouse. We discovered that Taf1 deletion in males was embryonically lethal, which may explain why no human null-variants have been identified. In the brains of Taf1 heterozygous females, no differences were found in gross structure, overall expression, and protein localization, suggesting extreme skewed X-inactivation towards the non-mutant chromosome. Nevertheless, these female mice exhibited a significant increase in weight, weight with age, and reduced movement, suggesting a small subset of neurons has been negatively impacted by Taf1 loss. Finally, this new mouse may be a future platform for the development of TAF1 disease therapeutics.

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“…Figure 6 Graph of motor neuron counts in genomically humanized FUS knock-in mice.There is no difference in the number of sciatic motor neurons in the lumbar spinal cord of genomically humanized FUS knock-in mice compared to wild-type mice aged 18 months. These mice express the human FUS gene, including all introns, exons and 3 UTR, from the endogenous mouse Fus loci(Devoy et al, 2021). The graph shows mean average total motor neurons, normalized to the number of sections counted.…”

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confidence: 99%

Sectioning and Counting of Motor Neurons in the L3 to L6 Region of the Adult Mouse Spinal Cord

et al. 2022

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Histology is the study of the microscopic structure of tissues. This protocol permits the generation of frozen transverse sections of lumbar spinal cord regions L3 to L6. It enables counting of murine ventral horn lumbar motor neurons in a reproducible manner. Methods include spinal column dissection, hydraulic extrusion, and histological processing. The preparation for cryo-sectioning includes embedding lumbar spinal cord in optimal cutting temperature (OCT) medium. The correct orientation of the tissue is critical as calculating the amount of tissue to discard saved time overall. Specific details regarding section thickness and mounting are described. These requirements not only allow optimum coverage of specific regions but also ensure that no individual motor neuron was counted twice. The Nissl bodies of the motor neurons were stained using gallocyanin. The sections obtained are all of a comparable area and quality assurance is consistent. The specificity of the staining enables the scientist to identify and reliably quantify lumbar motor neurons.

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Generation and analysis of innovative genomically humanized knockin SOD1, TARDBP (TDP-43), and FUS mouse models

Cited by 7 publications

References 69 publications

A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

A humanized knock-inCol6a1mouse recapitulates a deep-intronic splice-activating variant

Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders

Sectioning and Counting of Motor Neurons in the L3 to L6 Region of the Adult Mouse Spinal Cord

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