High throughput method for identification of sequence tags

• US 20030143578A1
• Filed: 08/26/2002
• Published: 07/31/2003
• Est. Priority Date: 08/24/2001
• Status: Abandoned Application

• Current Assignee: Health Research Incorporated
• Original Assignee: Health Research Incorporated
• Inventors: Pruitt, Steven C., Mielnicki, Lawrence M.
• Application Number:
  US10/227,719
• Publication Number:
  US 20030143578A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07K 2319/60 containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
• CPC Class Codes: C12N 15/1051 Gene trapping, e.g. exon-, intron-, IRES-, signal sequence-trap cloning, trap vectors
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12N 15/1086 Preparation or screening of expression libraries, e.g. reporter assays
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters

Abstract

The present invention provides a method for rapid identification of sites of insertion of DNA in to a cellular chromosome. The present invention also provides a gene trap vector. In one embodiment, the method of the present invention comprises the steps of stably transfecting a population of cells with a gene-trap vector, identifying cells with a trapped gene, distributing sorted cells into a matrix format, pooling cells from the matrix into discrete pools, producing cDNA sequence tags from the trapped genes in the pooled cells, making concatamers for each pool, cloning and sequencing the concatamers, defining the sequence tag of each well in the matrix.

Claims

1. A method for identification of Sequence Tags from trapped genes, comprising the steps of:

a) providing a gene-trap vector comprising a splice donor, a type IIS restriction endonuclease cleavage site and either a splice donor or a polyadenylation site;

b) preparing mRNA from cells stably transfected with the gene-trap vector;

c) synthesizing a first and second cDNA strands from the mRNA;

d) digesting with restriction endonucleases including the type IIS restriction endonucleases to produce Assay Tags, wherein each Assay Tag comprises a Sequence Tag and a portion of the gene-trap vector;

f) concatenating the Assay Tags;

g) amplifying and sequencing the concatamers to identify the sequence of the Assay Tags and the Sequence Tags.

2. The method of claim 1, wherein the second cDNA strand is biotinylated.

3. The method of claim 1, wherein the type IIS restriction endonuclease is selected from the group consisting of BsgI, BpmI, BsmF1, MmeI and Fok1.

4. A method for rapid analysis of gene expression comprising the steps of:

a) providing a gene-trap vector comprising a splice acceptor, a sequence encoding a fluorescent reporter protein, a type IIS restriction endonuclease cleavage site, and a splice donor or a polyadenylation site wherein the fluorescent reporter protein directly or indirectly produces fluorescence;

b) stably transfecting a population of cells with the gene-trap vector encoding a fluorescent reporter protein;

c) sorting the population of stably transfected cells by FACS to identify cells that express the fluorescent reporter protein, wherein expression of the reporter protein is indicative of the expression of the trapped gene;

d) distributing and expanding the stably transfected cells expressing the trapped gene in wells provided in a matrix format, wherein each well represents one trapped gene;

e) pooling cells from discrete wells in the matrix to represent pooling in x y and z axis;

f) preparing mRNA from the pooled cells;

g) synthesizing a first cDNA strand from the mRNA followed by synthesis of a second cDNA strand, wherein the second cDNA strand is linked to an anchor molecule;

h) digesting with the type IIS restriction endonucleases to produce Assay Tags comprising a Sequence Tag from the trapped gene and a portion of the gene-trap vector;

i) concatenating the Assay Tags from each pool;

j) cloning and sequencing the concatamers from each pool;

k) identifying the location of a sequence tag within the matrix by its unique presence in the discrete pools.

5. The method of claim 4, wherein the reporter protein is the green fluorescent protein.

6. The method of claim 4, wherein the reporter protein is the enhanced green fluorescent protein.

7. The method of claim 4, wherein the recognition endonucleases are BmpI and BsgI.

8. The method of claim 4, wherein the second strand of cDNA is linked to an anchor molecule;

9. The method of claim 4, wherein the anchor molecule is biotin.

10. A method for rapid analysis of gene expression comprising the steps of:

a) providing a gene-trap vector comprising a splice acceptor, a sequence encoding a fluorescent reporter protein, a type IIS restriction endonuclease cleavage site, and a splice donor or a polyadenylation site wherein the fluorescent reporter protein directly or indirectly produces fluorescence;

b) stably transfecting a population of cells with the gene-trap vector encoding a fluorescent reporter protein;

c) sorting the population of stably transfected cells by FACS to identify cells that express the fluorescent reporter protein, wherein expression of the reporter protein is indicative of the expression of the trapped gene;

d) expanding the stably transfected cells;

e) preparing mRNA from the expanded cells;

f) synthesizing a first cDNA strand from the mRNA followed by synthesis of a second cDNA strand, wherein the second cDNA strand is linked to an anchor molecule;

g) digesting with restriction endonucleases, wherein one of the endonucleases is the type IIS restriction endonucleases to produce Assay Tags comprising a Sequence Tag from the trapped gene and a portion of the gene-trap vector;

h) concatenating the Assay Tags from each pool;

i) cloning and sequencing the concatamers to obtain the sequence of the sequence tags.

11. The method of claim 10, wherein the reporter protein is the green fluorescent protein.

12. The method of claim 10, wherein the reporter protein is the enhanced green fluorescent protein.

13. The method of claim 10, wherein the recognition endonucleases are BmpI and BsgI.

14. The method of claim 10, wherein the second strand of cDNA is linked to an anchor molecule;

15. The method of claim 10, wherein the anchor molecule is biotin.

16. A nucleic acid construct comprising in downstream sequence:

a splice acceptor, a type II restriction endonuclease cleavage site; and

a splice donor or a polyadelylation site to terminate transcription.

17. The nucleic acid construct of claim 16 comprising in downstream sequence:

a splice acceptor, a first type IIS restriction endonuclease cleavage site, termination codons in all three reading frames, an internal ribosome entry site, a promotorless coding sequence encoding a polypeptide providing a positive or negative selection traits, a polyadynylation signal to terminate transcription, a promoter, a selectable marker, a second type IIS restriction endonuclease cleavage site, and a splice donor.

18. The nucleic acid construct of claim 17 comprising in downstream sequence:

a recombinogenic sequence, a splice acceptor, a first type IIS restriction endonuclease cleavage site, termination codons in all three reading frames, an internal ribosome entry site, a promotorless coding sequence encoding a polypeptide providing a positive or negative selection traits, a polyadynylation signal to terminate transcription, an insulator sequence, a promoter, a recombinogenic sequence, a selectable marker, a second type IIS restriction endonuclease cleavage site, and a splice donor.

19. The nucleic acid construct of claim 17, wherein the first IIS restriction endonuclease cleavage site is BsgI and the second IIS restriction endonuclease cleavage site is BpmI.

22. A kit for rapid analysis of gene expression comprising:

a gene-trap vector of claim 16, a type IIS endonucleases and one or more primers of SEQ ID NO;

2-9.

20. The nucleic acid construct of claim 118, wherein the polypeptide providing a positive or negative selection trait is a fluorescent protein.

21. The nucleic acid construct of claim 20, wherein the fluorescent protein is enhanced green fluorescence protein.