University of Massachusetts

St. Jude Children's Research Hospital

Jeanne B Lawrence

Lisa L Hall

Robert H Singer

Carol Villnave Johnson

Jun Jiang

Yigong Xing

Sheldon Penman

Andre J Van Wijnen

Gary S Stein

Janet L Stein

Jane B Lian

Cong-Mei Zeng

Scott Hiebert

Shari Meyers

Lindsay Shopland

Methods and compositions for reducing expression of genes on Chromosome 21 ('Chr 21') by targeting an XIST transgene to the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) gene or a Regulator of calcineurin 1 (RCAN1) gene, and cells and transgenic animals comprising an XIST transgene inserted into a DYRK1A or RCAN1 allele, e.g., cells and animals trisomic for human Chr 21 and mouse Chr 16.

Dosage compensating transgenes and cells

The present invention features compositions and methods for introducing, into cells, nucleic acids whose expression results in chromosomal silencing. The nucleic acids are targeted to specific chromosomal regions where they subsequently reduce the expression of deleterious genes, or cause the death of deleterious cells. Where the nucleic acid sequence is a silencing sequence, it may encode an Xist RNA or other non-coding, silencing RNA. Accordingly, the present invention features, inter alia, nucleic acid constructs that include a transgene (e.g., a silencing sequence encoding an Xist RNA or other non-coding RNA that silences a segment of a chromosome); first and second sequences that direct insertion of the silencing sequence into a targeted chromosome; and, optionally, a selectable marker.

Nucleic acid silencing sequences

Improved methodologies for in-situ hybridization and non-isotopic detection of nucleic acid sequences are provided which offer major increases of resolution, sensitivity, and simplicity unavailable in previously known techniques. The methodology is able to detect even a single-copy of a specific nucleic acid of interest under controlled conditions regardless of whether these are DNA or RNA sequences; or whether the nucleic acid sequence of interest is localized in the chromosomes, nucleus, or cytoplasm of a cell. The methods employ a variety of non-isotopic labels and detection means for rapid and reliable assays. The invention is also provided in kit form for use in the clinical/diagnostic laboratory such that a relatively unskilled person can accurately and reproducibly detect even a single-copy of a specific nucleic acid of interest.

In-situ hybridization of single-copy and multiple-copy nucleic acid sequences

Nuclear matrix targeting peptides (NMTPs) capable of delivering a variety of therapeutic and diagnostic agents, such as transcription regulatory factors, to the nuclear matrix of a cell are disclosed.

Isolated nuclear matrix targeting peptide

Non-isotopic in-situ hybridization method for detection of nucleic acids

Disclosed are in situ hybridization methods for differentially detecting an RNA and the gene from which it was transcribed, while preserving the spatial relationship of the RNA transcript and the gene. Also disclosed are in situ hybridization methods for simultaneously detecting two alleles of the same gene in a single cell, while differentially detecting RNA transcribed each of the two alleles. Also disclosed are in situ hybridization methods for detecting normal and defective RNA splicing.

Detection of individual gene transcription and splicing

In situ hybridization methods for assessing, determining or observing the RNA produced by transcriptionally active genes. In one embodiment, the methods allow simultaneous observation of the gene and its transcripts in a spatially correlated manner. As the in situ hybridization methods have established their ability to maintain the targeted mRNA at the site of its transcription, it can be determined which genes are being expressed and the level of expression can be quantitated.

Detection of individual gene transcription

Improved methodologies for in-situ hybridization and detection of hybridized nucleic acid sequences in cell cultures and tissue sections are provided which offer an increase of speed, sensitivity, and simplicity unavailable in previously known techniques. The invention detects specific nucleic acids of interest, particularly RNA sequences, within cells and tissues utilizing DNA of a particular size as a probe to find those sequences which are held substantially in common between the cell or tissue and the probe. The cells are fixed preferably in paraformaldehyde and then hybridized using a hybridization fluid for not less than 10 minutes but not substantially more than 24 hours. A variety of identifying labels are attached to the probe which permit quick and rapid detection via measurement of radioactive isotope decay or by colorimetric detection of enzymatic reaction products. The invention is intended for use as a diagnostic kit in clinical/diagnostic laboratory testing facilities in that it permits a relatively unskilled person to accurately and reproducibly detect a few molecules of a specific nucleic acid of interest in-situ in 10 minutes.

Growing community of inventors

Mapleville, RI, United States of America

Jeanne B Lawrence