The University of British Columbia

British Columbia Cancer Agency Branch

Bc Cancer Agency

The Public Health Agency of Canada

Robert A Holt

Steven J Jones

Wyeth W Wasserman

Elizabeth M Simpson

Daniel Goldowitz

Vikramjit S Chopra

Cletus D'Souza

Elodie Portales-Casamar

Charles De Leeuw

Robert C Brunham

Heinz Feldmann

Angela R Brooks-Wilson

Rachel Roper

Christopher Upton

Robin Coope

Nathalie Bastien

Caroline Astell

Perseus I Missirlis

Govinda Sharma

Daniel Horspool

Frank Plummer

Yan Li

The present invention is a method for determining the identity of the epitopes recognized by T-cells. The method consists of expressing an encoded library of candidate epitope sequences in a recipient reporter cell capable of providing a detectable signal upon cytotoxic attack from a single cognate T-cell followed by contacting the reporter cells with T-cells of interest. The reporter cells with a single indicating cytotoxic attack from a T-cell are isolated and then analyzed by next-generation sequencing in order to identify the epitope sequences. Specifically disclosed is a method in which a library of candidate epitope-encoding nucleic acids are expressed in cells which feature a membrane-bound major histocompatibility complex (MHC) protein, said library produced by transfection of plasmids featuring both a nucleotide encoding the candidate epitope and a nucleotide encoding a FRET-based fluorescent protein cleaved by granzyme.

T-cell epitope identification

Isolated polynucleotides comprising a TNNT1 mini-promoters are provided. The mini-promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, gene therapy, etc.

TNNT1 mini-promoters

Isolated polynucleotides comprising a UGT8 mini-promoters are provided. The mini-promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, gene therapy, etc.

UGT8 mini-promoters

Isolated polynucleotides comprising a DCX mini-promoters are provided. The mini-promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, gene therapy, etc.

DCX mini-promoters

The invention provides a system and method for synthesizing polynucleotides by solid phase assembly oligonucleotide precursors, in accordance with the method, a polynucleotide is partitioned into an ordered set of subunits, wherein each subunit is assembled in a single reaction from a subset of oligonucleotide precursors that uniquely anneal together to produce the subunit. The subunits are then assembled to form the desired polynucleotide. An important feature of the invention is the selection of subunits that are free of undesired sequence elements, such as palindromes, repetitive sequences, and the like, which would result in more than one subunit product alter ligating a pool of oligonucleotide precursors.

Gene synthesis by convergent assembly of oligonucleotide subsets

Isolated polynucleotides comprising a PCP2 mini-promoter are provided. The mini-promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, gene therapy, etc.

PCP2 mini-promoters

Isolated polynucleotides comprising an S100B promoter are provided, where an S100B regulatory element is operably joined to an S100B basal promoter utilizing a non-native spacing between the promoter and regulatory elements. The promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

S100B mini-promoters

Isolated polynucleotides comprising an OLIG1 promoter are provided, where an OLIG1 regulatory element is operably joined to an OLIG1 basal promoter utilizing a non-native spacing between the promoter and regulatory elements. The promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

Olig1 mini-promoters

Isolated polynucleotides comprising a CLDN5 mini-promoter are provided. The mini-promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The mini-promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

CLDN5 mini-promoters

Isolated polynucleotides comprising an MKI67 mini-promoter are provided. The mini-promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The mini-promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

MK167 mini-promoters

Isolated polynucleotides comprising a PITX3 promoter are provided, where a PITX3 regulatory element is operably joined to a PITX3 basal promoter utilizing a non-native spacing between the promoter and regulatory elements. The promoter may be operably linked to an expressible sequence, e.g. reporter genes, genes encoding a polypeptide of interest, regulatory RNA sequences such as miRNA, siRNA, anti-sense RNA, etc., and the like. In some embodiments a cell comprising a stable integrant of an expression vector is provided, which may be integrated in the genome of the cell. The promoter may also be provided in a vector, for example in combination with an expressible sequence. The polynucleotides find use in a method of expressing a sequence of interest, e.g. for identifying or labeling cells, monitoring or tracking the expression of cells, etc.

PITX3 expression promoters

The invention provides, in part, the genomic sequence of a putative coronavirus, the SARS virus, and provides novel nucleic acid and amino acid sequences that may be used, for example, for the diagnosis, prophylaxis, or therapy of a variety of SARS virus related disorders.

SARS virus nucleotide and amino acid sequences and uses thereof

The invention provides methods, kits, and compositions comprising novel mutant loxP sites. Such sites are particularly useful for procedures requiring multiple site-specific recombination reactions, including deletions or insertions of multiple genes or other sequences in the same organism, staged insertions or deletions of genes of the same organism at different times, assembly of large polynucleotide constructs by serial site-specific recombination, and the like. In one aspect, compositions of the invention includes particular mutant spacer oligonucleotides of loxP recombination elements, the recombination elements themselves, and pairs of non-promiscuous mutant loxP sites.

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Vancouver, Canada

Robert A Holt