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Methods and compositions for translational profiling and molecular phenotyping

Inactive Publication Date: 2011-03-24
THE ROCKEFELLER UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0043]FIG. 31 illustrates that cocaine treatment increases the frequency of small amplitude GABAergic mIPSCs in BAC D1 striatonigral neurons. (a) Representative spontaneous mIPSCs traces from BAC D1 striatonigral neurons (expressing soluble eGFP under the D1 promoter) taken from mice treated for 15 days with saline or (b) cocaine (20 mg/kg/day). (c) Bar graph summary of mean mIPSC frequency showing a increase in BAC D1 striatonigral neuron mIPSCs frequency following cocaine treatment (Mann Whitney Rank Sum Test, p<0.05, saline median=0.82 Hz, n=22; cocaine median=1.03 Hz, n=26). (d) Bar graph summary showing that the number of small amplitude mIPSCs (<75 pA) in equal length records (7 min) increased in BAC D1 striatonigral neurons following cocaine treatment (t test, p<0.05, saline=79.4±8.2, n=22; cocaine=104.2±6.3, n=26). (e) Representative variance mean current plots from saline treated and cocaine treated BAC D1 neurons suggesting that the cocaine induced small amplitude events arise from synapses tha

Problems solved by technology

The cellular complexity of many tissues poses a challenge for those seeking to characterize gene expression at this level.
Gene expression studies on isolated cells have been limited by stresses introduced during cellular isolation procedures, the adaptations which occur upon the loss of tissue-intrinsic signals that control cellular physiology in vivo, and the technical challenges associated with reproducible mRNA purification from fixed tissue.

Method used

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  • Methods and compositions for translational profiling and molecular phenotyping
  • Methods and compositions for translational profiling and molecular phenotyping
  • Methods and compositions for translational profiling and molecular phenotyping

Examples

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example 1

Molecular Tagging of a Ribosomal Proteins

[0216]mRNAs translated into protein are at one point usually attached to a ribosome or a polyribosome complex (polysomes). Any tag, not limited to those named herein, fused to a ribosomal protein allows for isolation of bound mRNAS. eGFP fused to the N-terminus of the large subunit ribosomal protein L10a, hereafter eGFP-L10a, was utilized in this and ensuing examples, but is by no ways limiting to the tens, hundreds, thousands, or even more tag-protein combinations that can be utilized.

[0217]FIG. 2: (a) A schematic is presented to illustrate affinity purification of eGFP-tagged Polysomes (originating from the target cell population) using anti-GFP antibody-coated beads (Schematic in a). Fusions of Enhanced Green Fluorescent Protein (eGFP) with ribosomal proteins were screened for efficient incorporation into polysomes to provide a tag for all translated cellular mRNAs. (b): A BAC carrying the eGFP-L10a fusion protein was transfected into HEK2...

example 2

Isolation, Purification and Analysis of Ribosome-mRNA Complexes in vitro

[0219]Rapid immunoaffinity purification of polysomes was achieved from HEK293T cells transiently transfected with the eGFP-L10a transgene but not mock transfected cells. HEK293T cells transfected with eGFP-L10a were homogenized in lysis buffer. The solubilized and clarified lysate was loaded onto a linear density (20-50% w / w) gradient of sucrose and centrifuged for 2 hours at 4° C. using a Beckman SW41 rotor at 40,000 r.p.m. (200,000×g). 750 μl fractions were collected as absorbance at 254 nm was monitored with an ISCO UA-6 UV detector.

[0220]Immunoaffinity purification of polysomes from transfected cell cultures (in which approximately 30% of cells expressed eGFP-L10a) gave an approximate 10% overall co-purification of untagged ribosomal proteins and ribosomal RNA, and led to the recovery of only translated mRNAs (Table 3).

TABLE 3Total RNA yields from cultured cell BACarray purifications.HEK293T cells were trans...

example 3

Characterization and Analysis of BACarray Transgenic Mice

[0223]1) Overview

[0224]Comparative analysis of BACarray data can provide important mechanistic insights into complex biological systems. BACarray translational profiling permits comprehensive studies of translated mRNAs in genetically defined cell populations, and their responses to physiological perturbations. To establish the generality of this approach, BACarray translational profiles for twenty four distinct and diverse CNS cell populations are presented here. Identification of cell-specific and enriched transcripts, previously not identified in whole tissue microarray studies are provided as examples to illustrate the added value of comparative analysis of these large datasets. The BAC transgenic strategy has been applied (Heintz, 2004; Yang et al., 1997) to provide high resolution anatomical data and BAC vectors for the design of genetic studies of specific, morphologically defined cells in the CNS (Gong et al., 2003) (w...

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Abstract

Methods and compositions are provided for translational profiling and molecular phenotyping of specific tissues, cells and cell subtypes of interest. The methods provided herein facilitate the analysis of gene expression in the selected subset present within a heterogeneous sample.

Description

CROSS-REFERENCE[0001]This application claims the benefit of U.S. Provisional Application Nos. 61 / 036,049 filed Mar. 12, 2008, 61 / 036,058 filed Mar. 12, 2008, 61 / 070,327, filed Mar. 21, 2008, and 61 / 199,108 filed Nov. 12, 2008 which applications are incorporated herein by reference in their entirety.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH[0002]This invention was made with the support of the U.S. Government under Grant No: AG09464 awarded by the National Institute on Aging, MH074866 awarded by the National Institute of Mental Health, DA10044 and 5F32DA021487 awarded by the National Institute on Drug Abuse, 5UL1RR024143 awarded by the National Institutes of Health / National Center for Research Resources, and NS34696 awarded by the National Institute of Neurological Disorders and Stroke. The U.S. Government may have certain rights to the subject matter provided herein.BACKGROUND OF THE INVENTION[0003]A paradigm in the development of new diagnostics and therapies for human diseases a...

Claims

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Application Information

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IPC IPC(8): C40B30/04C40B40/08C40B30/00C12Q1/68C12N15/63C12N15/11
CPCC12N15/111C12N2320/12G01N33/5023G01N33/5058C12N15/62A01K67/0278A01K2217/206A01K2227/105A01K2267/01G01N2800/285
Inventor HEINTZ, NATHANIELGREENGARD, PAULHEIMAN, MYRIAMSCHAEFER, ANNEDOYLE, JOSEPH P.DOUGHERTY, JOSEPH D.
Owner THE ROCKEFELLER UNIV
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