Yale University

Molecular Staging Inc.

Other

New York University

Sherman M Weissman

Xinghua Pan

Roger S Lasken

Peter M Rabinovich

Yatindra Prashar

Erkut Bahceci

Marina E Komarovskaya

Girish N Nallur

Prakash Kulkarni

Francis Collins

Ashwani Sood

Dennis Pereira

David C Ward

Jon Goguen

Charles Meyer Radding

Efim Ilya Golub

Peter E Newburger

Miguel A Alonso

Subrahmanyam V Yerramilli

Fang Wang

Namadev Baskaran

Samuel Katz

Jon J Jonsson

Andrew A Welcher

David Keefe

Basil Rigas

Saul M Honigberg

Lin Liu

Methods of identifying DNase I Hyper-Resistant Sites (DHRS), or in board sense, highly compact chromatin and characterizing the DNA methylation status of DMRs such as CpG islands and CpG island shores are provided. The methods are particularly useful for analysis of genomic DNA from low quantities of cells, for example, less than 1,000 cells, less than 100 cells, less than 10 cells, or even one cell, and can be used to generate chromatin and methylation profiles. The downstream analyses include in parallel massive sequencing, microarray, PCR and Sanger sequencing, hybridization and other platforms. These methods can be used to generate chromatin and DNA methylation profiles in drug development, diagnostics, and therapeutic applications are also provided.

Methods for closed chromatin mapping and DNA methylation analysis for single cells

Compositions and methods of making cells using RNA, and cells made using the disclosed compositions and methods are also provided. In exemplary embodiments, RNA is transfected into cells to effect a molecular, biological, physiological, or histological change in the cells. In preferred embodiments, the RNA is prepared in vitro, more preferably using a DNA template according to the provided compositions and methods. Methods for treating or inhibiting a disorder or disease such cancer are also provided. The methods can include, for example, locally or systemically administering to the host an effective amount of one or more RNAs; or an effective amount of population of cells isolated from the subject or a syngeneic or histocompatible subject, contacted ex vivo with one or RNAs, and optionally expanded. The cells can be, for example, immune cells or stem cells.

Cells prepared by transient transfection and methods of use thereof

The invention relates to a method for single cell analysis of relative telomere length using multiplex pre-PCR followed by a qPCR (SCT-pqPCR).

Method for single cell analysis of telomere length using pre-amplification and multiple-copy reference sequence

RNA prepared by in vitro transcription using a polymerase chain reaction (PCR)-generated template can be introduced into a cell to modulate cell activity. This method is useful in de-differentiating somatic cells to pluripotent, multipotent, or unipotent cells; re-differentiating stem cells into differentiated cells; or reprogramming of somatic cells to modulate cell activities such as metabolism. Cells can also be transfected with inhibitory RNAs, such as small interfering RNA (siRNA) or micro RNA (miRNA), or combinations thereof to induce reprogramming of somatic cells. For example, target cells are isolated from a donor, contacted with one or more RNA's causing the cells to be de-differentiated, re-differentiated, or reprogrammed in vitro, and administered to a patient in need thereof. The resulting cells are useful for treating one or more symptoms of a variety of diseases and disorders, for organ regeneration, and for restoration of the immune system.

Immune cells modified by transient transfection of RNA

Methods for preparing cDNA libraries from single and low quantities of cells are disclosed. The methods are based on the principles of multi-strand displacement amplification or semi-random primed polymerase chain reaction. The methods typically include a step of reverse transcription and subsequent amplification of cDNA. The methods can be adapted for preparation of cDNA libraries that are representative of mRNA or whole RNA expressed by the cell or cells. The cDNA is suitable for sequencing or microarray analysis.

Methods for preparing cDNA from low quantities of cells

Compositions for transient but prolonged exogenous mRNA expression through the use of the transcription system of negative strand RNA viruses, and methods of use thereof are disclosed. In some embodiments, the system contains only RNAs and does not include any DNA molecules. The compositions typically include an RNA template unit (rTeUn) that includes a virus regulatory sequences operably linked to a coding sequence of interest. The rTeUn is typically transfected to a host cell's cytoplasm in the presence of virus expression system proteins that mediate replication of the rTeUn and transcription of the transgene. The rTeUn RNA bonded to viral proteins exhibits high resistance to degradation, prolonged duration of expression, and is free of viral genes. The compositions can be used to reprogram cell. For example, the compositions and methods can be used to redirected lymphocytes to target cancer cells, or to dedifferentiate somatic cells into induce pluripotent stem cells.

Compositions and methods for transient expression of recombinant RNA

Method of de-differentiating and re-differentiating somatic cells using RNA

A method of mRNA production for use in transfection is provided, that involves in vitro transcription of PCR generated templates. This RNA can efficiently transfect different kinds of cells. This approach results in increased efficiency (fidelity and productivity) of mRNA synthesis and is less time consuming because it does not require cloning, and also consequently eliminates the unwanted errors and effects related to RNA made on DNA templates obtained with cloning techniques. The results of transfection of RNAs demonstrate that RNA transfection can be very effective in cells that are exceedingly difficult to transfect efficiently with DNA constructs. The method can be used to deliver genes into cells not- or only poorly transfectable for DNA, in vitro and in vivo.

Transient transfection with RNA

An improved method for screening genomic, cDNA, or any DNA fragments in general is described. Novel adapters are ligated to the ends of DNA fragments from two different individuals or two different pools of individuals. The DNA fragment-adapter complexes are mixed, denatured and reannealed to form homohybrid and heterohybrid DNA duplexes, which are separated based on characteristics of the adapters. Sequence differences between heterohybrids can be revealed as mismatched base pairs in the heterohybrid DNA duplex. Mismatch base pairs are discovered using genome mismatch scanning techniques that use thymine glycosylases and related enzymes that capture DNA containing mismatched base pairs. The perfectly base paired DNA or DNA containing mismatched base pairs can be further separated into homohybrids and heterohybrids using novel adapters that allow physical capture of either heterohybrid or homohybrid DNA.

Methods for identifying genes associated with diseases or specific phenotypes

DNA containing nucleotide base mispairs can be isolated using a modified rolling circle amplification procedure. Specific Y-shaped adapters permit the selective circularization of these fragments with a complementary splint oligonucleotide. Rolling circle amplification is then carried out with a DNA polymerase. Rolling circle amplification can also be carried out using a mixture of DNA circles having different lengths. Genetic phase of linked DNA markers can be determined by selective amplification of one parental haplotype. DNA fragments can also be converted into a form that can be utilized as rolling circle amplification templates by ligation of hairpin forming adapters to the ends of the fragments. Two or more DNA polymerases can be used in a rolling circle amplification reaction. DNA polymerase III has special properties that improve rolling circle amplification.

Methods for selectively isolating DNA using rolling circle amplification

A general method is described for screening cDNAs, genes or genome segments to directly isolate and characterize sequences associated with particular phenotypes. In the case of the human genome, a simplification of the starting material is needed, and a specific method to generate highly polymorphic genome subsets for this purpose is presented. The general screening method identifies DNA sequences containing allele frequency differences when groups with dissimilar phenotypes are compared. The approach is based on mathematical principles of inequality. A change in the abundance ratio of homoduplexes of perfectly matched sequences to heteroduplexes of perfectly matched sequences, or, conversely, of mismatched homoduplexes to mismatched heteroduplexes, serves as an indicator of allele frequency difference.

Allele frequency differences method for phenotype cloning

The present invention is directed to an approach to identify changes in gene expression by selective amplification of 3&prime; fragments of double stranded cDNAs.

Analysis of gene expression by display of 3'-end fragments of CDNAS

Genomic or cDNA, or fragments and mixtures thereof, can be screened by generation of subsets and then subjecting the subsets to mismatch scanning procedures. Alternatively, DNA fragments can be generated by cutting with a restriction endonuclease that generates variable overhangs. For either of the above methods, Y-shaped adapters having a region of non-complementary single-stranded DNA at the end can be used. Heterohybrid DNA, containing one DNA strand derived from each of two different samples, or homohybrids, containing DNA strands from the same sample, can be selected. Adapters attached to the ends of the fragments are designed to allow the selective isolation of homohybrid or heterohybrid DNA.

Methods for reducing the complexity of DNA sequences

The present invention comprises a method to identify granulocytic cell genes that are differentially expressed upon exposure to a pathogen or in a sterile inflammatory disease by preparing a gene expression profile of a granulocytic cell population exposed to a pathogen or isolated from a subject having a sterile inflammatory disease and comparing that profile to a profile prepared from quiescent granulocytic cells. The present invention is particularly useful for identifying cytokine genes, genes encoding cell surface receptors and genes encoding intermediary signaling molecules. The invention also includes methods to identify a therapeutic agent that modulates the expression of at least one gene in a granulocytic population. Genes which are differentially expressed during neutrophil contact with a pathogen, such as a virulent bacteria, or that are differentially expressed in a subject having a sterile inflammatory disease are of particular importance.

Growing community of inventors

New Haven, CT, United States of America

Sherman M Weissman