Methods and compositions for translational profiling and molecular phenotyping

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.

1 . A method of identifying a co-regulated gene set comprising:
 a. determining translational profiles for a plurality of cell types that express a gene associated with myelination;   b. comparing the translational profiles to determine what additional genes are similarly regulated thereby identifying a co-regulated gene set involved in myelination.   
     
     
         2 . The method of  claim 1  wherein the gene associated with myelination is Mbp. 
     
     
         3 . The method of  claim 1  wherein the gene set comprises at least two genes selected from the group consisting of Plp1, Cnp, Mog, Mal and Mobp. 
     
     
         4 . A co-regulated gene set involved in myelination comprising at least two genes listed in Table 9. 
     
     
         5 . The gene set of  claim 4 , comprising at least one of Plp1, Cnp, Mog, Mal, or Mobp. 
     
     
         6 . A method of detecting one or more cell type-enriched genes not detectable by the whole-tissue microarray technique comprising:
 a. assessing a translational profile for a cell type;   b. comparing the translational profile to the results of a whole-tissue microarray; and   c. determining the presence of one or more genes in the translational profile that are not present in the results of a whole-tissue microarray,   thereby detecting one or more cell type-enriched genes that are not detectable by the whole-tissue microarray technique.   
     
     
         7 . The method of  claim 6  wherein greater than 20%, 30% or 40% of the genes enriched in that cell type are not detectable by the whole-tissue microarray technique. 
     
     
         8 . The method of  claim 6  wherein the cell type is selected from the group consisting of striatal cell, cerebellar cell, cortical cell, hypothalamic cell, hippocampal cell, brainstem cell, and a spinal cord cell. 
     
     
         9 . The method of  claim 6  wherein the assessing a translational profile involves the use of a microarray. 
     
     
         10 . A method of identifying medium spiny neuron-enriched mRNAs, the method comprising:
 a. expressing a ribosomal protein regulated by a regulatory region specific to a gene expressed in medium spiny neurons in an organism;   b. isolating complexes comprising the ribosomal protein associated with mRNAs;   c. identifying the mRNAs in the complexes; and   d. comparing the mRNAs to those identified from a reference sample.   
     
     
         11 . The method of  claim 10  wherein the medium spiny neuron-enriched mRNAs are differentially expressed. 
     
     
         12 . The method of  claim 10  wherein the regulatory region is specific to a gene expressed in striatonigral neurons. 
     
     
         13 . The method of  claim 10  wherein the regulatory region is specific to a gene expressed in striatopallidal neurons. 
     
     
         14 . The method of  claim 10  wherein the expression is mediated by a bacterial artificial chromosome. 
     
     
         15 . The method of  claim 10  wherein the regulatory region comprises regulatory sequences from a Drd1a or a Drd2 locus. 
     
     
         16 . The method of  claim 10  wherein the expressing comprises introducing a bacterial artificial chromosome into an organism. 
     
     
         17 . The method of  claim 10  wherein the medium spiny neuron is associated with a disease or disorder. 
     
     
         18 . The method of  claim 17  wherein the disease or disorder is any one of Parkinson's disease, addiction, attention deficit hyperactivity disorder, or Huntington's disease. 
     
     
         19 . The method of  claim 17  wherein the method is used in guiding the selection of candidate targets for the treatment of the disease or disorder. 
     
     
         20 . The method of  claim 10  wherein the ribosomal protein is L10a. 
     
     
         21 . The method of  claim 10  wherein the ribosomal protein does not directly bind mRNA. 
     
     
         22 . The method of  claim 10  further comprising expressing a detectable tag. 
     
     
         23 . The method of  claim 22  wherein the detectable tag is eGFP. 
     
     
         24 . The method of  claim 10  wherein the isolating is carried out without crosslinking reagents. 
     
     
         25 . The method of  claim 10  further comprising administering a small molecule, a drug, an antibody, a hybrid antibody, an antibody fragment, a siRNA, an antisense RNA, an aptamer, a protein, or a peptide to the neuron. 
     
     
         26 . The method of  claim 25  wherein the drug is cocaine. 
     
     
         27 . The method of  claim 10  wherein the mRNA is further disposed on a microarray. 
     
     
         28 . The method of  claim 10  wherein the regulatory region comprises regulatory sequences from the Drd1a locus. 
     
     
         29 . The method of  claim 28  wherein the translational profile comprises Tac1, Pdyn, Slc35d3, Zfp521, Ebf1, Stmn2, Gnb4, Nrxn1, Eya1, Isl1, Gng2, or Crym. 
     
     
         30 . A method of identifying motor neuron-enriched mRNAs, the method comprising:
 a. expressing a ribosomal protein regulated by a regulatory region specific to a gene expressed in motor neurons in an organism;   b. isolating complexes comprising the ribosomal protein associated with mRNAs;   c. identifying the mRNAs in the complexes; and   d. comparing the mRNAs to those identified from a reference sample.   
     
     
         31 . The method of  claim 16  wherein the regulatory region comprises regulatory sequences from a choline acetyltransferase (Chat) locus. 
     
     
         32 . The method of  claim 16  wherein the motor neuron is a brain stem motor neuron. 
     
     
         33 . The method of  claim 16  wherein the motor neuron is a spinal cord motor neuron. 
     
     
         34 . The method of  claim 16  wherein the motor neuron is an upper motor neuron. 
     
     
         35 . The method of  claim 16  wherein the enriched mRNAs comprise one or more of those listed in Table 19. 
     
     
         36 . A method for assessing the translational profile of a cerebellar cell, comprising:
 a. expressing a ribosomal protein regulated by a regulatory region specific to a cerebellar gene in the cell;   b. isolating at least one complex comprising the ribosomal protein associated with a mRNA from the cell; and   c. identifying the mRNA from the complex whereby generating a translational profile.   
     
     
         37 . The method of  claim 36  wherein the expressing comprises transfecting a cell or an organism with a bacterial artificial chromosome. 
     
     
         38 . The method of  claim 36  wherein the cerebellar cell is associated with a disease or disorder. 
     
     
         39 . The method of  claim 38  wherein the disease or disorder is ataxia. 
     
     
         40 . The method of  claim 38  wherein the method is used in guiding the selection of candidate targets for the treatment of the disease or disorder. 
     
     
         41 . The method of  claim 36  wherein the ribosomal protein is L10a. 
     
     
         42 . The method of  claim 36  wherein the ribosomal protein does not directly bind mRNA. 
     
     
         43 . The method of  claim 36  further comprising expressing a detectable tag. 
     
     
         44 . The method of  claim 43  wherein the detectable tag is eGFP. 
     
     
         45 . The method of  claim 36  wherein the isolating is carried out without crosslinking reagents. 
     
     
         46 . The method of  claim 36  wherein the cerebellar cell is a Purkinje cell. 
     
     
         47 . The method of  claim 36  wherein the cerebellar cell is a Bergmann glia. 
     
     
         48 . The method of  claim 36  further comprising administering a small molecule, a drug, an antibody, a hybrid antibody, an antibody fragment, a siRNA, an antisense RNA, an aptamer, a protein, or a peptide to the neuron. 
     
     
         49 . The method of  claim 36  wherein the mRNA is further disposed on a microarray. 
     
     
         50 . The method of  claim 36  wherein the regulatory region comprises regulatory sequences from the Pcp2 locus. 
     
     
         51 . The method of  claim 50  wherein the translational profile comprises any one or more genes listed in Table 22. 
     
     
         52 . The method of  claim 36  wherein the regulatory region comprises regulatory sequences from the Septin4 locus. 
     
     
         53 . The method of  claim 52  wherein the translational profile comprises any one or more genes listed in Table 23. 
     
     
         54 . A kit comprising a recombinant vector engineered to express a nucleic acid sequence encoding a ribosomal protein and a detectable tag operably linked to an endogenous regulatory region. 
     
     
         55 . The kit of  claim 54  wherein the detectable tag is eGFP. 
     
     
         56 . The kit of  claim 54  wherein the ribosomal protein is L10a. 
     
     
         57 . The kit of  claim 54  wherein the ribosomal protein is fused in-frame to the detectable tag. 
     
     
         58 . The kit of  claim 54  wherein the recombinant vector is a bacterial artificial chromosome. 
     
     
         59 . The kit of  claim 54  wherein the endogenous regulatory region comprises the 5′ and/or 3′ untranslated sequences of a gene of interest. 
     
     
         60 . The kit of  claim 59  wherein the gene of interest is expressed a cell selected from the group consisting of striatal cell, cerebellar cell, cortical cell, hypothalamic cell, hippocampal cell, brainstem cell, and spinal cord cell.