A point mutation in the extracellular domain of CD4 completely abolishes CD4 T cell development in C57BL/6 mouse

Wang, Huijie; Li, Saichao; Chao, Tianzhu; Wang, Xugang; Shi, Lei; Zhang, Lichen; Liang, Yinming; Qin, Zheng; Lu, Liaoxun

doi:10.1016/j.molimm.2017.09.015

Cited by 5 publications

(8 citation statements)

References 20 publications

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“…We confirmed the mutant phenotype was only co-segregating with a mutation on Sgpl1 gene via exon sequencing on four different mutant mice of the same colony and B6 control ( Figure 1A ). The same method has also been described in previous studies ( 21 , 22 ). This mutant line carries a missense mutation on Sgpl1 , and its SPL protein changed methionine to lysine at position 467 (Ref.…”

Section: Resultsmentioning

confidence: 99%

“…Forward genetic screening can facilitate the robust and unbiased identification of novel genes or alleles contributing to the function of immune cells ( 20 ). We performed ENU mutagenesis to identify genetic factors regulating T cell development and function in mice ( 21 , 22 ). In the course of this work, we were surprised to discover that mice bearing the C57BL/6- Sgpl1 M467K Gris mutation in the Sgpl1 locus (or Sgpl1 M467K as abbreviation), exhibited an acute severe cutaneous inflammation phenotype under IMQ treatment while also being dramatically longer-lived than the previously reported Sgpl1 knockout mutant.…”

Section: Introductionmentioning

confidence: 99%

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A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

et al. 2022

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Sphingosine-1-phosphate lyase is encoded by the Sgpl1 gene, degrades S1P, and is crucial for S1P homeostasis in animal models and humans. S1P lyase deficient patients suffer from adrenal insufficiency, severe lymphopenia, and skin disorders. In this study, we used random mutagenesis screening to identify a mouse line carrying a missense mutation of Sgpl1 (M467K). This mutation caused similar pathologies as Sgpl1 knock-out mice in multiple organs, but greatly preserved its lifespan, which M467K mutation mice look normal under SPF conditions for over 40 weeks, in contrast, the knock-out mice live no more than 6 weeks. When treated with Imiquimod, Sgpl1M467K mice experienced exacerbated skin inflammation, as revealed by aggravated acanthosis and orthokeratotic hyperkeratosis. We also demonstrated that the IL17a producing Vγ6+ cell was enriched in Sgpl1M467K skin and caused severe pathology after imiquimod treatment. Interestingly, hyperchromic plaque occurred in the mutant mice one month after Imiquimod treatment but not in the controls, which resembled the skin disorder found in Sgpl1 deficient patients. Therefore, our results demonstrate that Sgpl1M467K point mutation mice successfully modeled a human disease after being treated with Imiquimod. We also revealed a major subset of γδT cells in the skin, IL17 secreting Vγ6 T cells were augmented by Sgpl1 deficiency and led to skin pathology. Therefore, we have, for the first time, linked the IL17a and γδT cells to SPL insufficiency.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

et al. 2022

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“…Herein, we performed a functional study of a mutant mouse line using YUMM1.7 Rosa26‐ZsGreen knock‐in melanoma cells. A novel point mutation of Cd11b was isolated from random N ‐ethyl‐ N ‐nitrosourea mutagenesis on the C57BL/6 background 30,31 . Interestingly, this mutation hits a highly conserved β‐propeller domain consisting of seven FG‐GAP repeats, with a serine to proline change at position 492 located in repeat 5 (Figure 4a, b).…”

Section: Resultsmentioning

confidence: 99%

A novel ZsGreen knock‐in melanoma cell line reveals the function of CD11b in tumor phagocytosis

et al. 2022

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Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) is an efficient tool for establishing genetic models including cellular models, and has facilitated unprecedented advancements in biomedical research. In both patients and cancer animal models, immune cells infiltrate the tumor microenvironment and some of them migrate to draining lymph nodes to exert antitumor effects. Among these immune cells, phagocytes such as macrophages and dendritic cells engulf tumor antigens prior to their crosstalk with T cells and elicit adaptive immune response against tumors. Melanoma cells are frequently used as a tumor model because of their relatively high level of somatic mutations and antigenicity. However, few genetic models have been developed using melanoma cell lines to track tumor cell phagocytosis, which is essential for understanding protective immune response in vivo. In this study, we used CRISPR/Cas9-mediated DNA cleavage and homologous recombination to develop a novel knock-in tool which expresses the ultra-bright fluorescent probe ZsGreen in YUMM1.7 melanoma cells. Using this novel tool, we measured the macrophagic engulfment of melanoma cells inside the tumor microenvironment. We also found that in tumor-grafted mice, a subset of dendritic cells efficiently engulfed YUMM1.7 cells and was preferentially trafficking tumor antigens to draining lymph nodes. In addition, we used this knock-in tool to assess the impact of a point mutation of CD11b on phagocytosis in the tumor microenvironment.Our results demonstrate that the ZsGreen-expressing YUMM1.7 melanoma model provides a valuable tool for the study of phagocytosis in vivo.

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“…T lymphocytes are pivotal components in the immune system of mammals to fight against pathogens and tumors and their development is under sophisticated molecular control which requires genetic dissection (Liang et al, 2013; Wang et al, 2017). In both CD4 and CD8 T cells, which are the two major subsets of T lymphocytes, CD44 expression on the cell surface is used as important marker to monitor T cell activation (Huet et al, 1989; DeGrendele et al, 1997).…”

Section: Resultsmentioning

confidence: 99%

Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice

et al. 2019

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It is a tempting goal to identify causative genes underlying phenotypic differences among inbred strains of mice, which is a huge reservoir of genetic resources to understand mammalian pathophysiology. In particular, the wild-derived mouse strains harbor enormous genetic variations that have been acquired during evolutionary divergence over 100s of 1000s of years. However, validating the genetic variation in non-classical strains was extremely difficult, until the advent of CRISPR/Cas9 genome editing tools. In this study, we first describe a T cell phenotype in both wild-derived PWD/PhJ parental mice and F1 hybrids, from a cross to C57BL/6 (B6) mice, and we isolate a genetic locus on Chr2, using linkage mapping and chromosome substitution mice. Importantly, we validate the identification of the functional gene controlling this T cell phenotype, Cd44 , by allele specific knockout of the PWD copy, leaving the B6 copy completely intact. Our experiments using F1 mice with a dominant phenotype, allowed rapid validation of candidate genes by designing sgRNA PAM sequences that only target the DNA of the PWD genome. We obtained 10 animals derived from B6 eggs fertilized with PWD sperm cells which were subjected to microinjection of CRISPR/Cas9 gene targeting machinery. In the newborns of F1 hybrids, 80% ( n = 10) had allele specific knockout of the candidate gene Cd44 of PWD origin, and no mice showed mistargeting of the B6 copy. In the resultant allele-specific knockout F1 mice, we observe full recovery of T cell phenotype. Therefore, our study provided a precise and rapid approach to functionally validate genes that could facilitate gene discovery in classic mouse genetics. More importantly, as we succeeded in genetic manipulation of mice, allele specific knockout could provide the possibility to inactivate disease alleles while keeping the normal allele of the gene intact in human cells.

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A point mutation in the extracellular domain of CD4 completely abolishes CD4 T cell development in C57BL/6 mouse

Cited by 5 publications

References 20 publications

A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

A Murine Point Mutation of Sgpl1 Skin Is Enriched With Vγ6 IL17-Producing Cell and Revealed With Hyperpigmentation After Imiquimod Treatment

A novel ZsGreen knock‐in melanoma cell line reveals the function of CD11b in tumor phagocytosis

Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice

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