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Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo.

ABSTRACT: We report the targeted mutagenesis of the murine iron regulatory protein (IRP)-1 and IRP2 genes, respectively, with a classical gene trap construct. Insertion of the targeting cassette into the second intron of either gene by homologous recombination interrupts their open reading frames near the N termini. Mice that are homozygous for the correctly modified IRP1 or IRP2 alleles, respectively, display a strong reduction (90%, IRP1(-/-)) or nondetectable levels (IRP2(-/-)) of the targeted proteins. Interestingly, the pre-mRNAs transcribed from the identical targeting cassettes are processed differently within the two different contexts. Detailed analysis of the respective products identifies the choice of alternative splice and 3' end processing sites in the same tissues in vivo. We discuss the implications for the understanding of RNA processing and for targeting strategies for functional genomics in the mouse.

SUBMITTER: Galy B

PROVIDER: S-EPMC1370593 | biostudies-literature | 2004 Jul

REPOSITORIES: biostudies-literature

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Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo.

Galy Bruno B Ferring Dunja D Benesova Monika M Benes Vladimir V Hentze Matthias W MW

RNA (New York, N.Y.) 20040701 7

We report the targeted mutagenesis of the murine iron regulatory protein (IRP)-1 and IRP2 genes, respectively, with a classical gene trap construct. Insertion of the targeting cassette into the second intron of either gene by homologous recombination interrupts their open reading frames near the N termini. Mice that are homozygous for the correctly modified IRP1 or IRP2 alleles, respectively, display a strong reduction (90%, IRP1(-/-)) or nondetectable levels (IRP2(-/-)) of the targeted proteins ...[more]

PMID: 15208438

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Project description:Purpose: Next-generation sequencing (NGS) has been utilized for systems-based analysis of all liver samples. The goals of this study are to use NGS-derived mouse CAR and human CAR initiated transcriptome profiling (RNA-seq) and find out similarity and difference drug processing gene (DPG) pattern after CAR activation in different genotype include WT (C57BL/6 and human CAR transgenic mice with C57BL/6 background) Methods: Liver mRNA profiles of wild-type (WT) and human CAR knockin (hCAR-TG) mice at the age of day 5 and day 60 treated with mouse CAR activator (TCPOBOP) and human CAR activator (CITCO) respectively were generated by deep sequencing, in triplicate, using HiSeq 2000 sequencer. The sequence reads that passed quality filters were analyzed at the transcript level with followed method: HISAT followed by Cufflinks. Results: Using an optimized data analysis workflow,RNA-Seq generated approximately 47 to 68 million reads per sample, among which approximately 40 to 60 million reads were uniquely mapped to the mouse reference genome (NCBI GRCm/38/mm10). And we identified 393 drug processing genes in the livers of WT and hCAR-TG with with HISAT workflow. RNA-seq data confirmed that among all the 393 DPGs with known important functions in xenobiotic biotransformation, 90 DPGs were not expressed in livers of any groups (threshold: average FPKM < 1 in all treatment groups); whereas a total of 303 genes were expressed in livers of at least one groups, among which 258 DPGs were differentially regulated by mCAR or hCAR activation in either Day 5 or Day 60 (FDR-BH<0.05), and 45 genes were stably expressed among all treatment groups. Conclusions: Our study represents the first detailed analysis of drug processing genes, with 3 biologic replicates, generated by RNA-seq technology. The optimized data analysis reported here should provide a framework for comparative investigations of expression profiles by mouse CAR activation and human CAR activation. Our results show that NGS offers a comprehensive and accurate quantitative and qualitative evaluation of mRNA content within tissues.

Dataset Information

Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo.

Publications

Targeted mutagenesis of the murine IRP1 and IRP2 genes reveals context-dependent RNA processing differences in vivo.

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