Spatiotemporal expression profile of no29/nucleophosmin3 in the intestine of Xenopus laevis during metamorphosis

Motoi, Natsuki; Hasebe, Takashi; Suzuki, Ken; Ishizuya‐Oka, Atsuko

doi:10.1007/s00441-011-1163-0

Cited by 5 publications

(9 citation statements)

References 43 publications

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“…We subsequently tried EGFP knock-in at endogenous gene loci in X. laevis embryos as a model of vertebrates, because gene knock-in in frogs including X. laevis has not yet been achieved, although targeted mutagenesis can be performed efficiently using TALENs 20 21 . Thus, we first targeted the no29 locus, one of the histone chaperone paralogues 22 in X. laevis , using TAL-PITCh ( Fig. 3a ).…”

Section: Resultsmentioning

confidence: 99%

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

et al. 2014

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Genome engineering using programmable nucleases enables homologous recombination (HR)-mediated gene knock-in. However, the labour used to construct targeting vectors containing homology arms and difficulties in inducing HR in some cell type and organisms represent technical hurdles for the application of HR-mediated knock-in technology. Here, we introduce an alternative strategy for gene knock-in using transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) mediated by microhomology-mediated end-joining, termed the PITCh (Precise Integration into Target Chromosome) system. TALEN-mediated PITCh, termed TAL-PITCh, enables efficient integration of exogenous donor DNA in human cells and animals, including silkworms and frogs. We further demonstrate that CRISPR/Cas9-mediated PITCh, termed CRIS-PITCh, can be applied in human cells without carrying the plasmid backbone sequence. Thus, our PITCh-ing strategies will be useful for a variety of applications, not only in cultured cells, but also in various organisms, including invertebrates and vertebrates.

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Section: Resultsmentioning

confidence: 99%

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

et al. 2014

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“…To test whether low homology paralogues can be simultaneously mutated by two different pairs of TALENs, we introduced two pairs of TALENs that separately target exon 2 of both histone chaperone paralogues, no29 and npm3 (Motoi et al . ). We could not design a single pair of TALENs that recognize both paralogues because their coding regions are short and have polymorphisms.…”

Section: Resultsmentioning

confidence: 97%

“…TALEN target sites for the no29 and npm3 genes were designed using the predicted exon 2 sequences from National Center for Biotechnology Information (NCBI) Refseq (Motoi et al . ; accession numbers, and , respectively). After linearization of plasmids by Sma I, mRNAs were synthesized using mMessage mMachine T7 Ultra Kit (Life Technologies, Carlsbad, CA, USA) as described previously (Suzuki et al .…”

Section: Methodsmentioning

confidence: 99%

Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator‐like effector nucleases

et al. 2013

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Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X. laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X. laevis.

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“…Common mature EC markers included genes relating to nutritional digestion and absorption, such as FABP6 , MT1A , FABP1 , FABP2 , APOA1 , and APOA4 [ 12 , 13 ], and the glutathione metabolism signaling gene ANPEP [ 24 ]. Immature ECs were characterized by the concerted expression of marker genes FABP3 , PCNA , MCM4 , and NPM , indicating distinct stages of maturation [ 14 , 25 ]. Well-known cell-type markers for progenitor cell (ProgCs) such as MKI67 , TOP2A , CENPF , and UBE2C [ 26 ] and PCNA and MCM6 for transient-amplifying cells (TACs) [ 27 ] were used to identify respective clusters.…”

Section: Resultsmentioning

confidence: 99%

Ileum tissue single-cell mRNA sequencing elucidates the cellular architecture of pathophysiological changes associated with weaning in piglets

et al. 2022

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Background In mammals, transitioning from sole milk uptake to the intake of solid feed results in dramatic developmental changes in intestinal function and immunological status. In fact, weaning stress is often accompanied by intestinal inflammatory processes. To develop effective intervention strategies, it is necessary to characterize the developmental pattern and immune response that occurs on weaning, as we have done in this study for piglets. Results To comprehensively delineate cell heterogeneity in ileum tissues and the underlying mechanisms in weaning-induced intestinal inflammation of piglets, we have analyzed the transcriptomes of 42,149 cells from ileum mucosa of normally suckling and post-weaned piglets. There were 31 cell subtypes including epithelial, stromal, and immune cells. A bifurcating trajectory was inferred to separate secretory and absorptive lineages. Integrated cross-species datasets showed well-conserved cellular architectures and transcription signatures between human and pig. Comparative analyses of cellular components, cell–cell communications, and molecular states revealed that T cell subpopulations were significantly altered in weaned piglets. We found that T helper (Th) 17 functional plasticity across changes in the cytokine milieu and the enrichment of granzyme B (GZMB)-expressing cytotoxic T cells potentially exacerbate mucosal inflammation via mitochondrial dysfunction in epithelial cells. Conclusions Our work has elucidated the single-cell molecular characteristics of the piglet ileum before and after weaning. We have provided an atlas that portrays the landscape of the intestinal pathophysiological inflammatory process associated with weaning, finding a level of conservation between human and pig that support the use of piglets as a model for human infants.

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Spatiotemporal expression profile of no29/nucleophosmin3 in the intestine of Xenopus laevis during metamorphosis

Cited by 5 publications

References 43 publications

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Microhomology-mediated end-joining-dependent integration of donor DNA in cells and animals using TALENs and CRISPR/Cas9

Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator‐like effector nucleases

Ileum tissue single-cell mRNA sequencing elucidates the cellular architecture of pathophysiological changes associated with weaning in piglets

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