A scalable two-step genome editing strategy for generating full-length gene humanized mice at diverse genomic loci
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PROVIDER: PRJNA1250549 | ENA |
REPOSITORIES: ENA
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Project description:Full-length gene-humanized mice, generated by replacing mouse loci with their human counterparts including untranslated and regulatory regions, offer a powerful in vivo platform to study human gene function. However, reliable humanization of large genomic regions remains challenging due to limitations in DNA insert size, complex protocols, and reliance on specialized materials. Here, we present a streamlined approach that enables full-length gene humanization through two sequential CRISPR-assisted homologous recombination steps in embryonic stem cells. This method supports targeted knock-in of genomic fragments exceeding 200 kbp and is applicable across multiple mouse strains. Humanized alleles generated using this strategy recapitulate human-like splicing isoforms and organ-specific gene expression, and restore essential functions in hematopoiesis, spermatogenesis, and survival. Furthermore, disease-associated mutations can be introduced into humanized alleles to generate in vivo models of human genetic disorders. This versatile platform enables creation of physiologically relevant, fully gene-humanized mouse models for broad applications in biomedical research.
2025-08-01 | GSE294590 | GEO
Project description:This study applies targeted Cas9-based gene insertion strategies for the correction of full-length dystrophin in a pre-clinical humanized mouse model of Duchenne muscular dystrophy. Following intramuscular or intravenous delivery, full-length dystrophin is restored in skeletal and cardiac muscle.
2021-04-24 | GSE173224 | GEO
Project description:Background: Hepatocytes are crucial for drug screening, disease modeling, and clinical transplantation, yet generating large numbers of hepatocytes in vitro faces challenges, particularly with traditional direct reprogramming methods. These methods often result in cells with limited functionality and residual memory of their original state. To address these issues, we have a novel two-step lineage reprogramming strategy, which converts fibroblasts into human induced hepatocytes (hiHeps) via an intermediate hepatic progenitor-like cell stage. hiHeps resembled closely with primary human hepatocytes in molecular and functional identity, showing great potential for hepatocyte-based application, however the mechanism underlying the two-step reprogramming is unclear. Methods: This study employs high-throughput single-cell RNA sequencing (scRNA-seq) to explore the molecular and cellular dynamics of this process. Besides, we applied fluorescence-activated cell sorting, RT-qPCR, and shRNA knockdown to validate the key findings of the scRNA-seq results. Results: The findings reveal that the two-step reprogramming mimics a trajectory of liver development, with similar transcriptional waves of developmental genes during reprogramming process. CD24 and DLK1 were identified as markers for two different hepatic progenitor populations, both contributing to hepatocyte maturation. Notably, Lipid metabolism was found to enhance the maturation of progenitors into functional hepatocytes. Additionally, HNF4A and HHEX play roles in determining cell fate between hepatocytes and intestinal cells in the late reprogramming stages. Conclusions: This research provides a comprehensive single-cell roadmap for generating hiHeps in vitro, enhancing our understanding and optimization of the two-step reprogramming protocol.
2025-04-16 | GSE278249 | GEO
Project description:IntroductionWhole-metagenome sequencing can be a rich source of information about the structure and function of entire metagenomic communities, but getting accurate and reliable results from these datasets can be challenging. Analysis of these datasets is founded on the mapping of sequencing reads onto known genomic regions from known organisms, but short reads will often map equally well to multiple regions, and to multiple reference organisms. Assembling metagenomic datasets prior to mapping can generate much longer and more precisely mappable sequences but the presence of closely related organisms and highly conserved regions makes metagenomic assembly challenging, and some regions of particular interest can assemble poorly. One solution to these problems is to use specialised tools, such as Kelpie, that can accurately extract and assemble full-length sequences for defined genomic regions from whole-metagenome datasets.MethodsKelpie is a kMer-based tool that generates full-length amplicon-like sequences from whole-metagenome datasets. It takes a pair of primer sequences and a set of metagenomic reads, and uses a combination of kMer filtering, error correction and assembly techniques to construct sets of full-length inter-primer sequences.ResultsThe effectiveness of Kelpie is demonstrated here through the extraction and assembly of full-length ribosomal marker gene regions, as this allows comparisons with conventional amplicon sequencing and published metagenomic benchmarks. The results show that the Kelpie-generated sequences and community profiles closely match those produced by amplicon sequencing, down to low abundance levels, and running Kelpie on the synthetic CAMI metagenomic benchmarking datasets shows similar high levels of both precision and recall.ConclusionsKelpie can be thought of as being somewhat like an in-silico PCR tool, taking a primer pair and producing the resulting 'amplicons' from a whole-metagenome dataset. Marker regions from the 16S rRNA gene were used here as an example because this allowed the overall accuracy of Kelpie to be evaluated through comparisons with other datasets, approaches and benchmarks. Kelpie is not limited to this application though, and can be used to extract and assemble any genomic region present in a whole metagenome dataset, as long as it is bound by a pairs of highly conserved primer sequences.
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2020-09-09 | GSE157660 | GEO
Project description:Bacterial expression of full length beta-amyloid (Abeta) is problematic because of toxicity and poor solubility of the expressed protein, and a strong tendency of Met35 to become oxidized in inclusion bodies. We have developed a semisynthetic method in which Abeta1-29 is expressed in bacteria as part of a fusion protein with a C-terminal intein and Chitin-Binding Domain (CBD). There is also a single residue, N-terminal Met extension. The protein, Met-Abeta1-29-Intein-CBD, is well expressed and highly water-soluble. After binding of the expressed protein to Chitin beads, treatment with sodium 2-mercapto-ethane sulfonate (MESNA) yields Met-Abeta1-29-MESNA, with a C-terminal thioester suitable for native chemical ligation. Met-Abeta1-29-MESNA is first subjected to CNBr cleavage, which removes the N-terminal Met residue, but leaves the thioester intact. We synthesized NH2-A30C-Abeta30-40, which has an N-terminal Cys residue and is the partner for native chemical ligation with Met-Abeta1-29-MESNA. Native chemical ligation proceeds rapidly and efficiently (>90% yield) to give A30C-Abeta1-40. The final step is selective desulfurization using Raney-Ni, which also proceeds rapidly and efficiently (>90% yield) to give native sequence Abeta1-40. Overall, this system is highly efficient, and can yield approximately 8-10 mg of pure Abeta1-40 from one liter of bacterial culture medium. This procedure is adaptable for producing other Abeta peptides. We have also expressed an Abeta construct bearing a point mutation associated with one type of familial Alzheimer's Disease, the Iowa mutation, i.e., Met-D23N-Abeta1-29-Intein-CBD. Since expression of the intein-containing fusion protein is robust in minimal media as well as standard enriched media, this procedure also can be readily modified for incorporating 15N or 13C labels for NMR. Future work will also include extending this system to longer Abeta peptides, such as Abeta1-42.
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Project description:Cytochrome P450 2D6 (CYP2D6) is among the most important genes involved in drug metabolism. Specific variants are associated with changes in the enzyme's amount and activity. Multiple technologies exist to determine these variants, like the AmpliChip CYP450 test, Taqman qPCR, or Second-Generation Sequencing, however, sequence homology between cytochrome P450 genes and pseudogene CYP2D7 impairs reliable CYP2D6 genotyping, and variant phasing cannot accurately be determined using these assays. To circumvent this, we sequenced CYP2D6 using the Pacific Biosciences RSII and obtained high-quality, full-length, phased CYP2D6 sequences, enabling accurate variant calling and haplotyping of the entire gene-locus including exonic, intronic, and upstream and downstream regions. Unphased diplotypes (Roche AmpliChip CYP450 test) were confirmed for 24 of the 25 samples, including gene duplications. Cases with gene deletions required additional specific assays to resolve. In total, 61 unique variants were detected, including variants that had not previously been associated with specific haplotypes. To further aid genomic analysis using standard reference sequences, we have established an LOVD-powered CYP2D6 gene-variant database, and added all reference haplotypes and data reported here. We conclude that our CYP2D6 genotyping approach produces reliable CYP2D6 diplotypes and reveals information about additional variants, including phasing and copy-number variation.
| S-EPMC5324676 | biostudies-literature
Project description:Humanized NOG-EXL (huNOG-EXL) mice control P. murina infection significantly better than control NOG-EXL mice, as evidenced by markedly decrased lung fungal burdens at both 3 and 6 weeks post-infection. Human cytokine-generating CD4+ and CD8+ T cells in the lung and serum IgM antibody, which were specifically in responses to pneumocystis antigen, were detected in huNOG-EXL mice only. For humanized NOG-EXL mouse group at 6 weeks post-infection, the whole lung RNAseq analyses were performed and the data indicated that the lower lung fungal burden is associated with higher CD209 gene expression.
2020-12-24 | GSE159562 | GEO
Project description:Single-Cell Roadmap of generating human induced hepatocyte-like cells from fibroblasts using two-step lineage reprogramming strategy
| PRJNA1166183 | ENA