University of California

Other

Columbia University

Envoy Therapeutics, Inc.

Tito A Serafini

Timothy F Kennedy

Marc Tessier-Lavigne

Thomas M Jessell

Jane Dodd

John J Ngai

Marysia Placzek

Andrew W Shyjan

David W Lin

Nathaniel Heintz

Yuan Zhang

David Leonardo

Percy Luu

Anne Shyjan

Candace Swimmer

Merysia Placzek

The invention provides methods for isolating cell-type specific mRNAs by selectively isolating ribosomes or proteins that bind mRNA in a cell type specific manner, and, thereby, the mRNA hound to the ribosomes or proteins that bind mRNA. Ribosomes, which are riboprotein complexes, bind mRNA that is being actively translated in cells. According to the methods of the invention, cells are engineered to express a molecularly tagged ribosomal protein or protein that binds mRNA by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding a ribosomal protein or protein that binds mRNA fused to a nucleotide sequence encoding a peptide tag. The tagged ribosome or mRNA binding protein can then be isolated, along with the mRNA bound to the tagged ribosome or mRNA binding protein, and the mRNA isolated and further used for gene expression analysis. The methods of the invention facilitate the analysis and quantification of gene expression in the selected cell type present within a heterogeneous cell mixture, without the need to isolate the cells of that cell type as a preliminary step.

Non human transgenic mammal comprising a transgene comprising a nucleotide sequence encoding a ribosomal protein fused to a peptide tag

The invention provides methods and compositions for isolation and assessment of riboprotein complexes.

Method for isolating cell type-specific mRNAs

Netrin proteins, nucleic acids which encode netrin proteins and hybridization reagents, probes and primers capable of hybridizing with netrin genes and methods for screening chemical libraries for lead compounds for pharmacological agents are provided.

Netrins

Nucleic acids are made by converting a primed single-stranded DNA to a double-stranded DNA by a method comprising the step contacting the single-stranded DNA with a DNA polymerase having 5&prime; exonuclease activity under conditions whereby the DNA polymerase converts the-single stranded DNA to the double-stranded DNA, wherein the single-stranded DNA is primed with oligonucleotide primer comprising a sequence complementary to the 3&prime; end of the single-stranded DNA, and at least one of the 5&prime; end of the primer and the single-stranded DNA comprises an RNA polymerase promoter joined to an upstream (5&prime;) flanking moiety which protects the promoter from the 5&prime; exonuclease activity of the DNA polymerase.

Methods for making nucleic acids

The invention provides methods and compositions for defining a cell type, generally involving the steps of (a) amplifying the mRNA of a single cell of a heterogenous population of cells; (b) probing a comprehensive expression library with the amplified mRNA to define a gross expression profile of the cell; and (c) comparing the gross expression profile of the cell with a gross expression profile of one or more other cells to define a unique expression profile of the cell, wherein the unique expression profile of the cell provides a marker defining the cell type

Methods for defining cell types

Specific netrin proteins, nucleic acids which encode netrin proteins and hybridization reagents, probes and primers capable of hybridizing with netrin genes and methods for screening chemical libraries for lead compounds for pharmacological agents useful in the diagnosis or treatment of disease associated undesirable cell growth are provided. An exemplary screen involves forming a mixture comprising a recombinant netrin protein, a natural intracellular netrin protein binding target, and a candidate pharmacological agent; incubating the mixture under conditions whereby, but for the presence of said candidate pharmacological agent, said netrin protein selectively binds said binding target; and detecting the presence or absence of specific binding of said netrin protein to said binding target.

Polynucleotide encoding human netrin-1

Nucleic acids are made by adding a known nucleotide sequence to the 3' end of a first RNA having a known sequence at the 5' end to form a second RNA and reverse transcribing the second RNA to form a cDNA. In one embodiment, the first RNA is an amplified mRNA, the known sequence at the 5' end comprises a poly(T) sequence, the adding step comprises using a polyadenyltransferase to add a poly(A) sequence to the 3' end, the reverse transcribing step is initiated at a duplex region comprising the poly(T) sequence hybridized to the poly(A) sequence, the cDNA is converted to double-stranded cDNA by a polymerase initiating from a noncovalently joined duplex region, and the double-stranded cDNA is transcribed to form one or more third RNAs.

The invention provides methods and compositions for defining a cell type, generally involving the steps of (a) amplifying the mRNA of a single cell of a heterogenous population of cells; (b) probing a comprehensive expression library with the amplified mRNA to define a gross expression profile of the cell; and (c) comparing the gross expression profile of the cell with a gross expression profile of one or more other cells to define a unique expression profile of the cell, wherein the unique expression profile of the cell provides a marker defining the cell type.

Method of defining cell types by probing comprehensive expression

Netrins, nucleic acids which encode netrin proteins and hybridization reagents, probes and primers capable of hybridizing with netrin genes and methods for screening chemical libraries for lead compounds for pharmacological agents are provided.

Human netrin-1

A novel classes of neural axon outgrowth promoting and orienting proteins, nucleic acids encoding such proteins and receptors which selectively bind such proteins are disclosed. The disclosed neural axon outgrowth promoting and orienting proteins include the laminin-related p75/p78 family; a family of vertebrate proteins which promote axon outgrowth and/or orientation, and p75/p78 family-specific receptors, including receptors found on spinal nerve axons, especially growth cones. Also disclosed are agents including peptides derived from the disclosed neural axon outgrowth promoting proteins capable of effecting axon outgrowth, orientation and regeneration. These agents provide small molecular weight modulators of nerve cell growth useful in the treatment of neurological disease and injury. The disclosed compositions also find use variously in screening chemical libraries for regulators of axon outgrowth and orientation, in genetic mapping, as probes for related genes, as diagnostic reagents for genetic neurological disease and in the production of specific cellular and animal systems for the development of neurological disease therapy.

Growing community of inventors

San Mateo, CA, United States of America

Tito A Serafini