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Engineering spacer specificity of the Cre/loxP system

ABSTRACT: Engineering spacer specificity of the Cre/loxP system

PROVIDER: PRJNA1070297 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR27756359_1.fastq.gz	Fastqsanger.gz
	SRR27756359_2.fastq.gz	Fastqsanger.gz
	SRR27756360_1.fastq.gz	Fastqsanger.gz
	SRR27756360_2.fastq.gz	Fastqsanger.gz
	SRR27756361_1.fastq.gz	Fastqsanger.gz

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Engineering spacer specificity of the Cre/loxP system

Project description:Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing how Cre distinguishes between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6,000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the flexibility of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations elucidated the mechanism behind this specificity switch. These findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications.

2024-07-25 | GSE254392 | GEO

Study on the genomic changes in ColIa2 Cre x RBP-Jkappa loxp/loxp mice

Project description:Skin tumor formed in the ColIa2 Cre x RBP-Jkappa loxp/loxp mice was analyzed for genomic changes by comparing with normal tissues from the same mice.

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Cre-loxP-induced Ewsr1-Atf1 translocation tumors

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Study on the genomic changes in ColIa2 Cre x RBP-Jkappa loxp/loxp mice

Project description:Skin tumor formed in the ColIa2 Cre x RBP-Jkappa loxp/loxp mice was analyzed for genomic changes by comparing with normal tissues from the same mice. Genomic DNA were isolated from each individual tumor and compared with the DNA extracted from the brain tissue in the same mouse.

2012-04-30 | E-GEOD-36464 | biostudies-arrayexpress

RNA Sequencing of mouse E15.5 Six2-Cre Irx3/5(loxP/loxP) whole kidneys

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RNAseq from mouse Cre-loxP-induced Ewsr1-Atf1 translocation

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Differential gene expression analysis for BAL cells isolated from LysM Cre and Netrin1loxp/loxp LysM Cre mice after intratracheal LPS challenge

Project description:Purpose: The goal of this study is to compare NGS-derived transcriptome profiling (mRNA-seq) of expressed genes between LysM Cre and Netrin1loxp/loxp LysM Cre mice 3 days after intratracheal LPS challenge in order to explain the worsened outcomes and increased levels of inflammation we measure in the Netrin1loxp/loxp LysM Cre mice Methods: Three days after mice were treated with intratracheal injections of LPS, BAL cells were collected and then depleted for neutrophils using antibody-mediated depletion. The remaining cells were used for RNA isolation. mRNA profiles were generated by deep sequencing, in quadruplicate (one sample in the Netrin1loxp/loxp LysM Cre group was excluded as the sequence depth was only half compare to other 3 replicates), using paired-end 75-cycle sequencing on an Illumina NextSeq 550 System. Bases with quality scores < 20 and adapter sequences were removed from raw data with Cutadapt (v1.15), followed by alignment of clean RNA-seq reads to GRCm38 with STAR(v2.5.3a). Gene abundance was counted by HTseq-count uniquely-mapped reads number with default parameter using GencodeM15. Genes with > 5 reads in at least one sample were included for differential expression analysis by DESeq2 software.

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Transcription profiling of prostate tumors derived from the PSA-Cre;PtenLoxP/LoxP mouse prostate cancer model

Project description:1. Comparison of gene expression profiles of normal prostates, hyperplastic prostates (4-5 month old mice) and prostate tumors (>10 month old mice) isolated from PSA-Cre;Pten-loxP/loxP mice 2.Comparison of the gene expression profiles and molecular subtyping of prostate tumors isolated from targeted Pten knockout mice

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Viral Cre-LoxP tools aid genome engineering in mammalian cells.

Project description:Background:Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. Results:We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. Conclusions:We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

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Changes in carbohydrate metabolism in alveolar epithelial type II cells in Ub-Cre and Hif1a loxp/loxp Ub cre mice in respnse to injurious mechanical ventilation (IMV)

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