ABSTRACT
Efficient incorporation of novel DNA sequences into a specific site in the genome of living human cells remains a challenge despite its potential utility to genetic medicine, biotechnology, and basic research. We find that a precisely placed double-strand break induced by engineered zinc finger nucleases (ZFNs) can stimulate integration of long DNA stretches into a predetermined genomic location, resulting in high-efficiency site-specific gene addition. Using an extrachromosomal DNA donor carrying a 12-bp tag, a 900-bp ORF, or a 1.5-kb promoter-transcription unit flanked by locus-specific homology arms, we find targeted integration frequencies of 15%, 6%, and 5%, respectively, within 72 h of treatment, and with no selection for the desired event. Importantly, we find that the integration event occurs in a homology-directed manner and leads to the accurate reconstruction of the donor-specified genotype at the endogenous chromosomal locus, and hence presumably results from synthesis-dependent strand annealing repair of the break using the donor DNA as a template. This site-specific gene addition occurs with no measurable increase in the rate of random integration. Remarkably, we also find that ZFNs can drive the addition of an 8-kb sequence carrying three distinct promoter-transcription units into an endogenous locus at a frequency of 6%, also in the absence of any selection. These data reveal the surprising versatility of the specialized polymerase machinery involved in double-strand break repair, illuminate a powerful approach to mammalian cell engineering, and open the possibility of ZFN-driven gene addition therapy for human genetic disease.
Subject(s)
Deoxyribonucleases/genetics , Gene Targeting/methods , Gene Transfer Techniques , Genetic Engineering/methods , Genome, Human/genetics , Zinc Fingers/genetics , Base Sequence , Evaluation Studies as Topic , Humans , Molecular Sequence DataABSTRACT
There are two functional insulin genes in the mouse genome. The Ins2 gene is imprinted and expressed monoallelically from the paternal allele in the yolk sac. In the present study we have re-examined the imprinting status of Ins1. We found that Ins1 is not expressed in the yolk sac of several laboratory mouse strains. The asynchrony of replication at the wild type locus was significantly lower than at imprinted loci and was more similar to non-imprinted loci. Finally, we have taken the advantage of the Ins1(neo) allele created by homologous recombination to examine the allelic usage at this locus. We observed that the neo gene inserted at the Ins1 locus was expressed from both the paternally and the maternally transmitted allele. Therefore, the Ins1 gene does not share any of the basic properties of imprinted genes. On the basis of these data, we concluded that Ins1 locus is unlikely to be imprinted in common laboratory mice.
Subject(s)
Genomic Imprinting , Insulin/genetics , Alleles , Animals , Fetus/metabolism , Genes, Reporter , Insulin/metabolism , Mice , Recombination, Genetic , Species Specificity , Yolk Sac/metabolismABSTRACT
Epigenetic control of transcription is essential for mammalian development and its deregulation causes human disease. For example, loss of proper imprinting control at the IGF2-H19 domain is a hallmark of cancer and Beckwith-Wiedemann syndrome, with no targeted therapeutic approaches available. To address this deficiency, we engineered zinc-finger transcription proteins (ZFPs) that specifically activate or repress the IGF2 and H19 genes in a domain-dependent manner. Importantly, we used these ZFPs successfully to reactivate the transcriptionally silent IGF2 and H19 alleles, thus overriding the natural mechanism of imprinting and validating an entirely novel avenue for 'transcription therapy' of human disease.
Subject(s)
Genetic Therapy/methods , Genomic Imprinting , Insulin-Like Growth Factor II/genetics , Neoplasms/therapy , Zinc Fingers , Beckwith-Wiedemann Syndrome/therapy , Female , Gene Expression Regulation , Gene Targeting/methods , Genes, Tumor Suppressor , Genetic Engineering , Humans , Kidney Neoplasms/therapy , Male , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/genetics , Wilms Tumor/therapyABSTRACT
The H19 and insulin-like growth factor 2 (Igf2) genes in the mouse are models for genomic imprinting during development. The genes are located only 90 kb apart in the same transcriptional orientation [1], but are reciprocally imprinted: Igf2 is paternally expressed while H19 is maternally expressed. It has been suggested that expression of H19 and repression of Igf2 (or the converse) on a given chromosome are mechanistically linked and that the parental imprint operates at the level of transcription [2]. Although expression of Igf2 and H19 is thought to be monoallelic, the data have so far been obtained exclusively by looking at steady-state RNA levels using techniques that reflect the average activity of the genes in a cell population [3] [4]. Here, we have adapted a fluorescent in situ hybridisation (FISH) method to detect nascent RNA molecules of Igf2 and H19 at the initial transcription sites in the nuclei of wild-type mouse embryonic liver cells. Nine different transcription patterns were observed, reflecting a high heterogeneity of transcription at the single-cell level. Our observations suggest that regulation of Igf2 and H19 by parental imprinting is much more complex than previously proposed and acts at both transcriptional and post-transcriptional levels.
