Patent Publication Number: US-9845481-B2

Title: Synthetic regulation of gene expression

Description:
This application is a continuation application of U.S. application Ser. No. 13/783,450 filed Mar. 4, 2013, which claims the benefit of U.S. Provisional Application No. 61/607,312, filed Mar. 6, 2012, both of which are incorporated herein by reference in their entirety for any purpose. 
    
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
     This invention was made with government support under Federal Grant Nos. IF32CA142095 and R01CA127727, both awarded by the National Institutes of Health: National Cancer Institute. The government has certain rights in the invention. 
    
    
     BACKGROUND 
     Current work in synthetic biology is focused on modifying protein structure and function, and regulation of gene expression circuits at the level of transcription promoters (Kwok, 2010). While promoter control of expression may be sufficiently specific in bacterial systems, heterologous promoters confer only limited control of gene expression (e.g. cell-specific expression) in eukaryotic cells, possibly because of a lack of correct chromatin assembly (Wolffe, 1999). The limited control of gene expression in eukaryotic cells by heterologous promoters alone may be insufficient for certain therapies and other biotechnological applications. Complete spatiotemporal control of gene expression is important for the use of at least a subset of biological molecules in gene therapy and other biotechnological applications. 
     SUMMARY 
     In some embodiments, synthetic regulatory constructs are provided. In some embodiments, a synthetic regulatory construct comprises a cell-specific promoter, a heterologous gene, and a cell-specific exon. In some embodiments, the cell-specific exon is excluded in the cell in which the cell-specific promoter is most active. In some embodiments, inclusion of the exon results in the product of the heterologous gene being inactive, or no product of the heterologous gene being produced. In some embodiments, inclusion of the exon results in a frame shift in the heterologous gene or a stop codon before or within the heterologous gene, or both. 
     In some embodiments, the cell-specific exon is included in the cell in which the cell-specific promoter is most active. In some embodiments, exclusion of the exon results in the product of the heterologous gene being inactive, or no product of the heterologous gene being produced. In some embodiments, exclusion of the exon results in a frame shift in the heterologous gene or a stop codon before or within the heterologous gene, or both. 
     In some embodiments, a synthetic regulatory construct comprises a cell-specific RNA stability element. In some such embodiments, the cell-specific RNA stability element is a microRNA target sequence. 
     In some embodiments, a cell-specific promoter is at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold more active in a selected cell than in at least one other cell comprised in the same organism as the selected cell. In some embodiments, a cell-specific promoter is at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold more active in a selected cell than in at least two, at least three, or at least five other cells comprised in the same organism as the selected cell. In some embodiments, a cell-specific promoter is at least 2-fold, at least 3-fold, at least 5-fold, at least 7-fold, or at least 10-fold more active in a selected cell than in any other cell comprised in the same organism as the selected cell. 
     In some embodiments, a cell is an animal cell. In some embodiments, a cell is a mammalian cell. In some embodiments, the mammal is selected from human, mouse, rat, dog, chimpanzee, and monkey. In some embodiments, a cell is a plant cell. In some embodiments, a plant is selected from a monocot and a dicot. In some embodiments, a cell is a fungal cell. 
     In some embodiments, cells comprising synthetic regulatory constructs are provided. In some embodiments, a cell is an animal cell. In some embodiments, a cell is a mammalian cell. In some embodiments, a cell is comprised in a mammal. In some embodiments, a cell is ex vivo or in vitro. In some embodiments, a cell is a plant cell. In some embodiments, a cell is comprised in a plant. In some embodiments, a cell is a fungal cell. 
     In some embodiments, methods of expressing a heterologous gene in a selected cell are provided. In some embodiments, a method comprises introducing a synthetic regulatory construct into the selected cell. In some embodiments, a cell is an animal cell. In some embodiments, a cell is a mammalian cell. In some embodiments, the mammal is selected from human, mouse, rat, dog, chimpanzee, and monkey. In some embodiments, a selected cell is selected from mesenchymal, epithelial, neuronal, heart muscle, skeletal muscle, smooth muscle, and embryonic muscle cells. In some embodiments, a cell is comprised in a mammal. In some embodiments, a cell is a plant cell. In some embodiments, a cell is comprised in a plant. In some embodiments, a cell is a fungal cell. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1  shows in  FIG. 1A  schematic diagrams of four plasmids with various elements for synthetic regulation of firefly luciferase expression, and in  FIG. 1B  expression of firefly luciferase in mesenchymal and epithelial cells transfected with the plasmids from  FIG. 1A , as described in Example 1. 
         FIG. 2  shows in  FIG. 2A  a schematic diagram of a plasmid designed for epithelial-cell specific expression of Cre recombinase (Cre), and the predicted spliced products and translation products resulting from that plasmid in mesenchymal and epithelial cells, and a second plasmid that expresses dsRED in the absence of Cre protein and EGFP in the presence of Cre protein;  FIG. 2B  shows fields of mesenchymal and epithelial cells transfected with the plasmids from  FIG. 2A , showing expression of dsRED (left panels) and EGFP (right panels); and  FIG. 2C  shows quantitation of EGFP expression in mesenchymal and epithelial cells transfected with the plasmids from  FIG. 2A  over time, as described in Example 2. 
         FIG. 3  shows in  FIG. 3A  a schematic diagram of a plasmid designed for epithelial cell-specific expression of diphtheria toxin, and the predicted spliced products and translation products resulting from that plasmid in mesenchymal and epithelial cells; and in  FIG. 3B  detection of transcripts resulting from the plasmid from  FIG. 3A  in transfected mesenchymal and epithelial cells, as described in Example 3. 
         FIG. 4  shows in  FIG. 4A  a schematic diagram of a plasmid designed for mesenchymal cell-specific expression of a protein x, and four predicted spliced products resulting from that plasmid in mesenchymal and epithelial cells; and in  FIG. 4B  detection of transcripts resulting from the plasmid from  FIG. 4A  in transfected mesenchymal and epithelial cells, as described in Example 4. 
     
    
    
     DETAILED DESCRIPTION 
     The present inventors have developed a synthetic gene regulation system that provides better spatiotemporal regulation than promoters used alone. In some embodiments, the synthetic gene regulation system described herein provides better cell-specificity than cell-specific promoters used alone. In some embodiments, the synthetic gene regulation system described herein provides better temporal regulation than certain promoters used alone. In some embodiments, the synthetic gene regulation system may be used for cell-specific and/or temporal regulation of expression of various agents, including, but not limited to, therapeutic agents. Accordingly, in some embodiments, artificial control of the expression of biological molecules, including, but not limited to, therapeutic biological molecules, via combinations of cell-specific promoters, cell-specific exons and cell-specific RNA stability elements, is provided. In some embodiments, artificial control of the expression of biological molecules, including, but not limited to, therapeutic biological molecules, via combinations of temporally-regulated promoters, temporally-regulated exons and temporally-regulated RNA stability elements, is provided. In some embodiments, such modular regulatory elements may come from different genes or be artificial combinations of known sub-elements. 
     As described herein, post-transcriptional regulation offers additional control of gene expression, particularly in eukaryotic cells. For example, in some embodiments, alternative cassette exons may either be included or skipped in eukaryotic mRNA in a cell-specific manner (“cell-specific exons”). In some embodiments, alternative exon inclusion may disrupt the expression of, or change the function of, a gene product. Like transcription promoters, exons are recognized by regulatory macromolecular complexes in the cell. Further, this recognition may be modular: exons may be moved into heterologous contexts and still be recognized by the cell. 
     As a further example, in some embodiments, post-transcriptional regulation can determine the stability of mRNAs in a cell-specific manner (“cell-specific RNA stability elements”). In some embodiments, by affecting RNA stability, such post-transcriptional regulators may determine RNA levels. This regulation may also be accomplished by an independent set of macromolecular complexes. In some embodiments, regulation of RNA stability involves small non-coding RNAs known as microRNAs (miRNAs). In some embodiments, mRNAs may be regulated at the level of translation efficiency, for example, by proteins and miRNAs, which may exert their function via signals in the 5′ and/or 3′ untranslated regions (UTRs) of the messenger. 
     As discussed herein, in some embodiments, the various levels of regulation (including, but not limited to, transcription, alternative splicing, and RNA stability) are orthogonal and thus provide independent modes that in combination provide multiplied specificity. Thus, in some embodiments, by combining modular control elements from different genes, novel or artificially stringent patterns of gene expression can be engineered. Currently, such modular control does not appear to be appreciated in synthetic biology. 
     Definitions 
     The subject matter disclosed herein is described using several definitions, as set forth below and throughout the application. 
     Unless otherwise noted, the terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. In addition to the definitions of terms provided below, it is to be understood that as used in the specification, embodiments, and in the claims, “a”, “an”, and “the” can mean one or more, depending upon the context in which it is used. 
     As used herein, “about,” “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” or “approximately” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term. 
     As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” 
     As used herein, the term “cell-specific promoter” refers to a promoter that is at least 3-fold more active in a selected cell than in one or more other cells. In some embodiments, a cell specific promoter is at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more active in a selected cell than in one or more other cells. In some embodiments, a cell specific promoter is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold more active in a selected cell than in other cells found in the same organism as the selected cell. In some embodiments, a selected cell is a particular cell type. 
     As used herein, the term “cell-specific exon” refers to an exon that is present in a transcript or absent from a transcript through alternative splicing at a rate that is at least 3-fold greater in a selected cell than in one or more other cells. In some embodiments, a cell-specific exon is present in a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is present in a transcript in one or more other cells. In some embodiments, a cell-specific exon is absent from a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is absent from a transcript in one or more other cells. In some embodiments, a cell-specific exon is present in a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is present in a transcript in other cells found in the same organism as the selected cell. In some embodiments, a cell-specific exon is absent from a transcript in a selected cell at a rate that is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is absent from a transcript in other cells found in the same organism as the selected cell. In some embodiments, a selected cell is a particular cell type. 
     In some embodiments, the rate that an exon is present is determined as the ratio of spliced transcript with the exon present to spliced transcript with the exon absent in a particular cell. In some embodiments, the rate an exon is absent is determined as the ratio of spliced transcript with the exon absent to spliced transcript with the exon present in a particular cell. As a nonlimiting example, the following table shows hypothetical rates that a hypothetical cell-specific exon A is present in a transcript in hypothetical cells x and y: 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                   
                 Relative amount 
                 Relative amount 
                   
                 Fold difference  
               
               
                   
                 of transcript 
                 of transcript  
                   
                 in rate of inclusion  
               
               
                   
                 with exon A 
                 with exon A  
                 Rate exon 
                 in cell x 
               
               
                   
                 included 
                 excluded 
                 is included 
                 versus cell y 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
            
               
                 Cell x 
                 10 
                 2 
                 5 
                 10-fold 
               
               
                 Cell y 
                 5 
                 10 
                 0.5 
                   
               
               
                   
               
            
           
         
       
     
     As used herein, the terms “exclusion”, “excluded”, and similar terms, when used in relation to an exon, mean that the rate that an exon is included in a transcript, as described above, is less than 1. Alternatively, the terms “exclusion”, “excluded”, and similar terms, when used in relation to an exon, mean that the rate that an exon is excluded from a transcript, determined in a similar manner as described above for inclusion of an exon (although with the numerator and denominator reversed), is greater than 1. 
     As used herein, the terms “inclusion”, “included”, and similar terms, when used in relation to an exon, mean that the rate that an exon is included in a transcript, as described above, is greater than 1. Alternatively, the terms “inclusion”, “included”, and similar terms, when used in relation to an exon, mean that the rate that an exon is excluded from a transcript, determined in a similar manner as described above for inclusion of an exon (although with the numerator and denominator reversed), is less than 1. 
     As used herein, the term “heterologous gene” refers to any gene or coding sequence that is not controlled in its natural state (e.g., within a non-genetically modified cell) by the cell-specific promoter to which it is operably linked in a particular construct, and whose gene, in its natural state, does not contain the cell-specific exon included in the particular construct. In some embodiments, the gene or coding sequence is described as being heterologous to the cell-specific promoter and/or heterologous to the cell-specific exon. 
     As used herein, the terms “cell-specific RNA stability element” and “cell-specific RNA stability element” refer to a regulatory element, which may be in the 3′-untranslated region or 5′-untranslated region of a transcript, that increases the stability or translation of a transcript in a selected cell and/or decreases the stability or translation of the transcript in one or more cells other than the selected cell, such that the stability or translation of the transcript in the selected cell is at least 2-fold greater than the stability or translation of the transcript in one or more other cells. In some embodiments, the stability or translation of the transcript in the selected cell is at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than in one or more other cells. In some embodiments, the stability or translation of the transcript in the selected cell is at least 2-fold, 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than in other cells found in the same organism as the selected cell. In some embodiments, a selected cell is a particular cell type. In some embodiments, an RNA stability element is an element that decreases the stability or translation of the transcript in one or more cells other than the selected cell. In some embodiments, a cell-specific RNA stability element is a microRNA target sequence. In some embodiments, a cell-specific RNA stability element is a binding site for an hnRNP protein. Nonlimiting exemplary hnRNPs include AUF-1, AUF-2, and HuR. 
     The term “microRNA target sequence” refers to a RNA stability element that comprises a seed match sequence for a particular microRNA. A seed match sequence is a sequence that is complementary to at least nucleotides 2 to 7 of the microRNA. In some embodiments, a microRNA target sequence comprises a sequence that is complementary to more of the microRNA than just nucleotides 2 to 7. In some embodiments, a microRNA target sequence decreases the stability or translation of a transcript in cells that express the particular microRNA. 
     Nonlimiting Exemplary Cell-Specific Promoters 
     Many cell-specific promoters are known in the art. Nonlimiting exemplary mammalian cell-specific promoters have been characterized and used in mice expressing Cre recombinase in a cell-specific manner. Certain nonlimiting exemplary mammalian cell-specific promoters are listed in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Nonlimiting exemplary cell-specific promoters 
               
            
           
           
               
               
               
               
               
               
            
               
                 Promoter 
                 Cell/tissue specificity 
                 Promoter 
                 Cell/tissue specificity 
                 Promoter 
                 Cell/tissue specificity 
               
               
                   
               
               
                 A930038C07Rik,  
                 cortex, striatum, and 
                 Col2a1, collagen,  
                 differentiating 
                 GZMB, granzyme B 
                 activated T cells 
               
               
                 RIKEN cDNA  
                 cerebellum 
                 type II, 
                 chondrocytes, notochord, 
                 (granzyme 2,  
                   
               
               
                 A930038C07  
                   
                 alpha 1 (mouse) 
                 submandibular glands 
                 cytotoxic T- 
                   
               
               
                 gene 
                   
                   
                   
                 lymphocyte- 
                   
               
               
                 (mouse) 
                   
                   
                   
                 associated 
                   
               
               
                   
                   
                   
                   
                 serine esterase  
                   
               
               
                   
                   
                   
                   
                 1) (human) 
                   
               
               
                 ACTA1, actin, 
                 adult striated muscle 
                 Cr2, complement  
                 Mature transitional B cells  
                 Grik4, glutamate  
                 area CA3 of the 
               
               
                 alpha 1, 
                 fibers and embryonic 
                 receptor 
                   
                 receptor, 
                 hippocampus 
               
               
                 skeletal muscle  
                 striated muscle cells of the 
                 2 (mouse) 
                   
                 ionotropic, kainate 4 
                   
               
               
                 (human) 
                 somites and heart 
                   
                   
                 (mouse) 
                   
               
               
                 Alb, albumin  
                 Hepatocytes (liver) 
                 Cspg4,  
                 NG2 expressing glial cells 
                 HBB, hemoglobin,  
                 Erythroid tissues 
               
               
                 (rat) 
                   
                 chondroitin  
                 and vasculature 
                 beta 
                   
               
               
                   
                   
                 sulfate 
                 throughout the brain as 
                 (human) 
                   
               
               
                   
                   
                 proteoglycan 4  
                 well as in NG2-expressing 
                   
                   
               
               
                   
                   
                 (mouse) 
                 cells in other tissues from 
                   
                   
               
               
                   
                   
                   
                 late embryonic stages 
                   
                   
               
               
                   
                   
                   
                 (~embryonic day 14) 
                   
                   
               
               
                   
                   
                   
                 throughout adulthood 
                   
                   
               
               
                 Alpl, alkaline  
                 embryonic primordial 
                 Ctgf, connective  
                 Cortex and hippocampus 
                 Hoxb7, homeobox B7 
                 mesonephric duct  
               
               
                 phosphatase, 
                 germ cells 
                 tissue 
                   
                 (mouse) 
                 and its developmental  
               
               
                 liver/bone/ 
                   
                 growth factor  
                   
                   
                 derivatives 
               
               
                 kidney  
                   
                 (mouse) 
                   
                   
                 (the Wolffian  
               
               
                 (mouse) 
                   
                   
                   
                   
                 duct, the 
               
               
                   
                   
                   
                   
                   
                 collecting duct  
               
               
                   
                   
                   
                   
                   
                 epithelium 
               
               
                   
                   
                   
                   
                   
                 of kidney and ureteral 
               
               
                   
                   
                   
                   
                   
                 epithelium),  
               
               
                   
                   
                   
                   
                   
                 ureteric bud 
               
               
                   
                   
                   
                   
                   
                 and all ureteric bud 
               
               
                   
                   
                   
                   
                   
                 epithelial cells;  
               
               
                   
                   
                   
                   
                   
                 low levels 
               
               
                   
                   
                   
                   
                   
                 of expression  
               
               
                   
                   
                   
                   
                   
                 in the dorsal 
               
               
                   
                   
                   
                   
                   
                 root ganglia and  
               
               
                   
                   
                   
                   
                   
                 the spinal 
               
               
                   
                   
                   
                   
                   
                 cord 
               
               
                 Amh, anti- 
                 testis Sertoli cells 
                 Cyp39a1,  
                 cortex, hippocampus, 
                 Ins2, insulin 2 (rat) 
                 Pancreatic beta cells 
               
               
                 Mullerian 
                   
                 cytochrome 
                 striatum, olfactory bulb 
                   
                   
               
               
                 hormone  
                   
                 P450, family 39,  
                 and cerebellum 
                   
                   
               
               
                 (mouse) 
                   
                 subfamily 
                   
                   
                   
               
               
                   
                   
                 a, polypeptide  
                   
                   
                   
               
               
                   
                   
                 1 (mouse) 
                   
                   
                   
               
               
                 Aqp2,  
                 kidney cells (collecting 
                 Ddx4, DEAD  
                 male and female germ 
                 Itgax, integrin  
                 Dendritic cells 
               
               
                 aquaporin 2 
                 duct) and testes (sperm) 
                 (Asp-Glu- 
                 cells starting at embryonic  
                 alpha X 
                   
               
               
                 (mouse) 
                   
                 Ala-Asp) box  
                 day (e)15-e18 
                 (mouse) 
                   
               
               
                   
                   
                 polypeptide 4 
                   
                   
                   
               
               
                   
                   
                 (mouse) 
                   
                   
                   
               
               
                 Atoh1, atonal  
                 neural progenitors of the 
                 Emx1, empty  
                 neurons of the neocortex 
                 KRT14, keratin 14  
                 skin, the oral ectoderm 
               
               
                 homolog 1 
                 cerebellar rhombic lip, 
                 spiracles 
                 and hippocampus 
                 (human) 
                 including the dental 
               
               
                 ( Drosophila )  
                 dorsal hindbrain and 
                 homolog 1  
                   
                   
                 lamina at 11.75  
               
               
                 (mouse) 
                 spinal cord, as well as in 
                 ( Drosophila ) 
                   
                   
                 d.p.c., and 
               
               
                   
                 inner-ear primordia (with 
                 (mouse) 
                   
                   
                 the dental  
               
               
                   
                 a limited amount of 
                   
                   
                   
                 epithelium by 
               
               
                   
                 ectopic expression in the 
                   
                   
                   
                 14.5 d.p.c. 
               
               
                   
                 primordium of the 
                   
                   
                   
                   
               
               
                   
                 hippocampus but not the 
                   
                   
                   
                   
               
               
                   
                 cortex) 
                   
                   
                   
                   
               
               
                 Camk2a, 
                 forebrain; pyramidal cell  
                 En1, engrailed  
                 Spinal cord V1 
                 Lck, lymphocyte  
                 thymocytes 
               
               
                 calcium/ 
                 layer 
                 1 (mouse)  
                 interneurons, the 
                 protein 
                   
               
               
                 calmodulin- 
                   
                   
                 embryonic 
                 tyrosine kinase  
                   
               
               
                 dependent  
                   
                   
                 mesencephalon and 
                 (mouse) 
                   
               
               
                 protein kinase 
                   
                   
                 rhombomere 1 by E9, as 
                   
                   
               
               
                 II alpha (mouse) 
                   
                   
                 well as in the ventral 
                   
                   
               
               
                   
                   
                   
                 ectoderm of the limbs, in a 
                   
                   
               
               
                   
                   
                   
                 subset of somite cells, and 
                   
                   
               
               
                   
                   
                   
                 some mesoderm-derived 
                   
                   
               
               
                   
                   
                   
                 tissues 
                   
                   
               
               
                 Cartpt,  
                 cortex, hippocampus, and 
                 En2, engrailed  
                 developing 
                 Lepr, leptin receptor 
                 hypothalmus (arcuate, 
               
               
                 CART 
                 cerebellum 
                 2 (mouse) 
                 mesencephalon, 
                 (mouse) 
                 dorsomedial,  
               
               
                 prepropeptide  
                   
                   
                 rhombomere 1, and jaw 
                   
                 lateral, and 
               
               
                 (mouse) 
                   
                   
                 muscles, as well as the 
                   
                 ventromedial nuclei), 
               
               
                   
                   
                   
                 embryonic and adult 
                   
                 limbic and  
               
               
                   
                   
                   
                 cerebellum 
                   
                 cortical brain 
               
               
                   
                   
                   
                   
                   
                 regions (basolateral 
               
               
                   
                   
                   
                   
                   
                 amygdaloid  
               
               
                   
                   
                   
                   
                   
                 nucleus, 
               
               
                   
                   
                   
                   
                   
                 piriform cortex,  
               
               
                   
                   
                   
                   
                   
                 and lateral 
               
               
                   
                   
                   
                   
                   
                 entorhinal  
               
               
                   
                   
                   
                   
                   
                 cortex), and 
               
               
                   
                   
                   
                   
                   
                 retrosplenial cortex 
               
               
                 Cd19, CD19  
                 B cells 
                 Eno2, enolase  
                 neurons in many tissue 
                 Lgr5, leucine  
                 crypt base  
               
               
                 antigen 
                   
                 2, gamma, 
                 types 
                 rich repeat 
                 columnar cells 
               
               
                 (mouse) 
                   
                 neuronal (rat) 
                   
                 containing G protein 
                 in small  
               
               
                   
                   
                   
                   
                 coupled receptor 5 
                 intestine (stem 
               
               
                   
                   
                   
                   
                 (mouse) 
                 cells of the small  
               
               
                   
                   
                   
                   
                   
                 intestine) 
               
               
                   
                   
                   
                   
                   
                 and colon 
               
               
                 Cdh5, cadherin  
                 Embryonic and adult 
                 Fabp4, fatty  
                 Adipose tissue 
                 Lyz2, lysozyme 2  
                 myeloid  
               
               
                 5 (mouse) 
                 expression in endothelium 
                 acid binding 
                   
                 (mouse) 
                 cells—including 
               
               
                   
                 of developing and 
                 protein 4,  
                   
                   
                 monocytes, mature 
               
               
                   
                 quiescent vessels of all 
                 adipocyte 
                   
                   
                 macrophages, and 
               
               
                   
                 organs examined, as well 
                 (mouse) 
                   
                   
                 granulocytes 
               
               
                   
                 as within a subset of 
                   
                   
                   
                   
               
               
                   
                 hematopoietic cells 
                   
                   
                   
                   
               
               
                 Cga,  
                 anterior and intermediate 
                 Foxg1, forkhead  
                 telencephalon, anterior 
                 Meox2, mesenchyme 
                 epiblast-derived  
               
               
                 glycoprotein 
                 lobes of the pituitary 
                 box G1 
                 optic vesicle (developing 
                 homeobox 2 (mouse) 
                 tissues as early as  
               
               
                 hormones,  
                 gland, as well as in cardiac 
                 (mouse) 
                 lens and retina), otic 
                   
                 embryonic day 5; 
               
               
                 alpha subunit 
                 and skeletal muscle; low 
                   
                 vesicle, facial and head 
                   
                 all primitive  
               
               
                 (mouse) 
                 to no level of expression is 
                   
                 ectoderm, olfactory 
                   
                 ectoderm by 
               
               
                   
                 detected in the posterior 
                   
                 epithelium, mid-hindbrain 
                   
                 embryonic day  
               
               
                   
                 pituitary, lungs, kidneys, 
                   
                 junction and pharyngeal 
                   
                 7 (but not 
               
               
                   
                 brain, adrenal gland and 
                   
                 pouches 
                   
                 endoderm or 
               
               
                   
                 gonads 
                   
                   
                   
                 trophectoderm) 
               
               
                 Chat, choline 
                 cholinergic neurons 
                 Foxp3, forkhead  
                 Cd4 + Cd25  high Cd127 low   T 
                 Mnx1, motor  
                 Motor neurons 
               
               
                 acetyltransferase  
                   
                 box P3 
                 cells from the lymph 
                 neuron and 
                   
               
               
                 (mouse) 
                   
                 (mouse) 
                 nodes, spleen and thymus 
                 pancreas homeobox  
                   
               
               
                   
                   
                   
                   
                 1 (mouse) 
                   
               
               
                 Ckm, creatine  
                 skeletal and cardiac 
                 GFAP, glial  
                 primarily in the central 
                 Mybpcl, myosin  
                 cortex and  
               
               
                 kinase, 
                 muscle 
                 fibrillary acidic 
                 nervous system, affecting 
                 binding 
                 hippocampus 
               
               
                 muscle  
                   
                 protein (human) 
                 astrocytes, 
                 protein C, slow-type 
                   
               
               
                 (mouse) 
                   
                   
                 oligodendroglia, 
                 (mouse) 
                   
               
               
                   
                   
                   
                 ependyma and some 
                   
                   
               
               
                   
                   
                   
                 neurons; also periportal 
                   
                   
               
               
                   
                   
                   
                 cells of the liver 
                   
                   
               
               
                 Myf5, myogenic  
                 Skeletal muscle and 
                 Myh11, myosin,  
                 vascular and nonvascular 
                 Myh6, myosin, heavy 
                 heart 
               
               
                 factor 5 
                 dermis 
                 heavy 
                 smooth muscle 
                 polypeptide 6, cardiac 
                   
               
               
                 (mouse) 
                   
                 polypeptide  
                   
                 muscle, alpha, murine 
                   
               
               
                   
                   
                 11, smooth 
                   
                 (murine) 
                   
               
               
                   
                   
                 muscle (mouse) 
                   
                   
                   
               
               
                 Nefl,  
                 Projection neurons 
                 Nes, nestin (rat) 
                 central and peripheral 
                 Neurog1, neurogenin 1 
                 cortex, hippocampus, 
               
               
                 neurofilament, 
                   
                   
                 nervous system by 
                 (mouse) 
                 thalamus, hypothalamus 
               
               
                 light 
                   
                   
                 embryonic day 11 and a 
                   
                 and the cochlear- 
               
               
                 polypeptide  
                   
                   
                 few isolated kidney and 
                   
                 vestibular ganglion 
               
               
                 (mouse) 
                   
                   
                 heart cells 
                   
                   
               
               
                 Neurog3,  
                 small intestine (base of 
                 Nkx2-1, NK2  
                 major subgroups of brain 
                 Nr5a1, nuclear  
                 in ventromedial 
               
               
                 neurogenin 3 
                 intestinal crypts) and fetal 
                 homeobox 1 
                 interneuron progenitors, 
                 receptor 
                 hypothalamic  
               
               
                 (rat) 
                 pancreatic epithelial cells 
                 (mouse) 
                 developing lung, thyroid, 
                 subfamily 5,  
                 nucleus, 
               
               
                   
                   
                   
                 and pituitary 
                 group A, 
                 cerebral cortex,  
               
               
                   
                   
                   
                   
                 member 1 (mouse) 
                 and a few scattered  
               
               
                   
                   
                   
                   
                   
                 cells in the 
               
               
                   
                   
                   
                   
                   
                 caudal brainstem,  
               
               
                   
                   
                   
                   
                   
                 as well 
               
               
                   
                   
                   
                   
                   
                 as in pituitary,  
               
               
                   
                   
                   
                   
                   
                 gonad, and 
               
               
                   
                   
                   
                   
                   
                 adrenal tissue 
               
               
                 Olfr151,  
                 olfactory sensory neurons 
                 Omp, olfactory  
                 mature olfactory sensory 
                 PTH, parathyroid  
                 Parathyroid tissue 
               
               
                 olfactory  
                   
                 marker 
                 neurons 
                 hormone 
                   
               
               
                 receptor  
                   
                 protein (mouse) 
                   
                 (human) 
                   
               
               
                 151 (mouse) 
                   
                   
                   
                   
                   
               
               
                 Pax3, paired  
                 dorsal neural tube and 
                 Pcp2, Purkinje  
                 Purkinje cells 
                 Pf4, platelet factor 4 
                 megakaryocytes 
               
               
                 box gene 3 
                 somites of embryonic day 
                 cell protein 
                   
                 (mouse) 
                   
               
               
                 (mouse) 
                 9-11.5 embryos and in the 
                 2 (L7) (mouse) 
                   
                   
                   
               
               
                   
                 cardiac neural crest cells 
                   
                   
                   
                   
               
               
                   
                 and colonic epithelia of 
                   
                   
                   
                   
               
               
                   
                 embryonic day 11.5 
                   
                   
                   
                   
               
               
                   
                 embryos 
                   
                   
                   
                   
               
               
                 Plp1,  
                 oligodendrocytes and 
                 Pomc, pro- 
                 arcuate nucleus of the 
                 Prm1, protamine 1 
                 Male germ line 
               
               
                 proteolipid  
                 Schwann cells 
                 opiomelanocortin- 
                 hypothalamus and nucleus 
                 (mouse) 
                   
               
               
                 protein 
                   
                 alpha 
                 of the solitary tract in the 
                   
                   
               
               
                 (myelin) 1  
                   
                 (mouse) 
                 hindbrain 
                   
                   
               
               
                 (mouse) 
                   
                   
                   
                   
                   
               
               
                 Prrx1, paired  
                 early limb bud 
                 Pvalb,  
                 most neurons that express 
                 Rbp3, retinol binding 
                 Photoreceptor cells 
               
               
                 related 
                 mesenchyme and in a 
                 parvalbumin 
                 parvalbumin including 
                 protein 3, interstitial  
                   
               
               
                 homeobox  
                 subset of craniofacial 
                 (mouse) 
                 interneurons in the brain 
                 (rat) 
                   
               
               
                 1 (rat) 
                 mesenchyme, some 
                   
                 and proprioceptive 
                   
                   
               
               
                   
                 female germline 
                   
                 afferent sensory neurons 
                   
                   
               
               
                   
                 expression 
                   
                 in the dorsal root ganglia 
                   
                   
               
               
                 Scnn1a, sodium  
                 cortex, striatum, 
                 Shh, sonic  
                 Distal posterior region of 
                 Sim1, single-minded 
                 paraventricular 
               
               
                 channel, 
                 hippocampus and 
                 hedgehog 
                 the limb buds of embryos 
                 homolog 1  
                 hypothalamus  
               
               
                 nonvoltage- 
                 cerebellum 
                 (mouse) 
                 aged embryonic day 10 to 
                 ( Drosophila ) 
                 and other 
               
               
                 gated 1 alpha 
                   
                   
                 12 
                 (mouse) 
                 parts of the brain 
               
               
                 (mouse) 
                   
                   
                   
                   
                   
               
               
                 Slc6a3,  
                 adult dopaminergic cell 
                 Togln, transgelin  
                 Vascular smooth muscle 
                 Tek, endothelial- 
                 female germline  
               
               
                 solute carrier 
                 groups (substantia nigra 
                 (mouse) 
                 cells 
                 specific 
                 as well as 
               
               
                 family 6  
                 (SN) and ventral tegmental 
                   
                   
                 receptor tyrosine  
                 tyrosine  
               
               
                 (neuro- 
                 area (VTA), as well as in 
                   
                   
                 kinase 
                 endothelial and 
               
               
                 transmitter 
                 the retrorubral field) 
                   
                   
                 (mouse) 
                 hematopoietic cells 
               
               
                 transporter,  
                   
                   
                   
                   
                   
               
               
                 dopamine), 
                   
                   
                   
                   
                   
               
               
                 member 3  
                   
                   
                   
                   
                   
               
               
                 (mouse) 
                   
                   
                   
                   
                   
               
               
                 Th, tyrosine  
                 Dopaminergic neurons 
                 Thy1, thymus cell  
                 neurons of the postnatal 
                 Vil1, villin 1 (mouse) 
                 Epithelial cells  
               
               
                 hydroxylase 
                   
                 antigen 1,  
                 cortex and hippocampus 
                   
                 of the small 
               
               
                 (rat) 
                   
                 theta (mouse) 
                   
                   
                 and large intestines 
               
               
                 Vsx2, visual  
                 retina and Muller  
                 Wap, whey acidic  
                 mammary gland tissues 
                 Wfs1, Wolfram  
                 cortex, hippocampus, 
               
               
                 system 
                 glial cells 
                 protein (mouse) 
                   
                 syndrome 
                 striatum, thalamus and 
               
               
                 homeobox  
                   
                   
                   
                 1 homolog (human) 
                 cerebellum 
               
               
                 2 (rat) 
                   
                   
                   
                 (mouse) 
                   
               
               
                 Wnt1, wingless- 
                 embryonic neural tube, 
                 dlx6a, distal-less 
                 GABAergic forebrain 
                 Vimentin 
                 Mesenchymal cells 
               
               
                 related 
                 midbrain, dorsal and 
                 homeobox gene  
                 neurons 
                   
                   
               
               
                 MMTV  
                 ventral midlines of the 
                 6a, Danio 
                   
                   
                   
               
               
                 integration 
                 midbrain and caudal 
                 rerio 
                   
                   
                   
               
               
                 site 1 (mouse) 
                 diencephalon, the mid- 
                   
                   
                   
                   
               
               
                   
                 hindbrain junction and 
                   
                   
                   
                   
               
               
                   
                 dorsal spinal cord 
                   
                   
                   
                   
               
               
                 E-cadherin 
                 Epithelial cells 
               
               
                   
               
            
           
         
       
     
     In some embodiments, a cell-specific promoter is a promoter that is active in plants. Many exemplary cell-specific plant promoters are known in the art. See, e.g., U.S. Pat. Nos. 5,097,025; 5,783,393; 5,880,330; 5,981,727; 7,557,264; 6,291,666; 7,132,526; and 7,323,622; and U.S. Publication Nos. 2010/0269226; 2007/0180580; 2005/0034192; and 2005/0086712, which are incorporated by reference herein in their entireties for any purpose. 
     Nonlimiting Exemplary Cell-Specific Exons 
     Many cell-specific exons are known in the art. Certain nonlimiting exemplary cell-specific exons are described in Table 2 and in the examples provided herein. The literature references provided in Table 2 are each incorporated by reference herein in their entireties for any purpose. 
     In some embodiments, a cell-specific exon is included in a selected cell. In some embodiments, the inclusion of the cell-specific exon allows for expression of an active product from a heterologous gene into which the exon is incorporated. In some embodiments, the inclusion of a cell-specific exon results in expression of an inactive product from a heterologous gene. In some embodiments, the inclusion of a cell-specific exon results in a decrease or elimination of expression of the heterologous gene product, for example, by inserting a stop codon upstream of the start codon of the heterologous gene and/or by frame-shifting the heterologous protein coding region. 
     In some embodiments, a cell-specific exon is excluded in a selected cell. In some embodiments, the exclusion of the cell-specific exon allows for expression of an active product from a heterologous gene into which the exon is incorporated. In some embodiments, the exclusion of a cell-specific exon results in expression of an inactive product from a heterologous gene. In some embodiments, the exclusion of a cell-specific exon results in a decrease or elimination of expression of the heterologous gene product, for example, by frame-shifting the heterologous gene coding region. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Nonlimiting exemplary cell-specific exons 
               
            
           
           
               
               
               
               
               
            
               
                   
                   
                 Tissue 
                 Exemplary RNA 
                   
               
               
                 Gene 
                 Exon 
                 specificity 
                 elements 
                 References 
               
               
                   
               
               
                 FGFR2 
                 IIIb 
                 Epithelial 
                 IAS2, ISAR, DICE 
                 Oltean et al, 
               
               
                   
                   
                   
                   
                 2008; Seth et al, 
               
               
                   
                   
                   
                   
                 2008 and ref 
               
               
                   
                   
                   
                   
                 therein 
               
               
                   
               
               
                 FGFR2 
                 IIIc 
                 Mesenchymal 
                 IAS2, ISAR, DICE 
                 Oltean et al, 
               
               
                   
                   
                   
                   
                 2008; Seth et al, 
               
               
                   
                   
                   
                   
                 2008 and ref 
               
               
                   
                   
                   
                   
                 therein 
               
               
                   
               
               
                 c-src 
                 N1 
                 Neuronal 
                 Fox2/KSRP, 
                 Reviewed in 
               
               
                   
                   
                   
                 PTB/nPTB sites 
                 Black, 2003. 
               
               
                   
                   
                   
                   
                 Originally 
               
               
                   
                   
                   
                   
                 identified in: 
               
               
                   
                   
                   
                   
                 Black, 1992; Chan 
               
               
                   
                   
                   
                   
                 and Black, 1995; 
               
               
                   
                   
                   
                   
                 Min et al, 1997; 
               
               
                   
                   
                   
                   
                 Chan and Black, 
               
               
                   
                   
                   
                   
                 1997 
               
               
                   
               
               
                 cardiac troponin 
                 Exon 5 
                 Embryonic 
                 Muscle specific 
                 Ladd et al., 2001 
               
               
                 T 
                   
                 muscle 
                 enhancers 
                 and ref therein 
               
               
                   
               
               
                 Smarcc2, Ptprf, 
                 Smarcc2 exon 16a, Ptprf 
                 Neuronal 
                 Nova-1 and 
                 Ule et al., 2006 
               
               
                 Brd9, Map4, 
                 exon 6a, Brd9 exon 5, 
                   
                 Nova-2 sites aka 
                   
               
               
                 Ank3 
                 Map4 exon 18, Ank3 exon 
                   
                 YCAY clusters 
                   
               
               
                   
                 31a 
                   
                   
                   
               
               
                   
               
               
                 Cardiac troponin 
                 Cardiac troponin T exon 5, 
                 Cardiac troponin 
                 CUGBP sites 
                 Reviewed in 
               
               
                 T, insulin 
                 insulin receptor exon 11, 
                 T in heart, rest 
                   
                 Ranum and 
               
               
                 receptor, muscle 
                 chloride channel intron 2 
                 in skeletal 
                   
                 Cooper, 2006. 
               
               
                 specific chlorine 
                 and exon 7a 
                 muscle 
                   
                 Originally 
               
               
                 channel, etc 
                   
                   
                   
                 identified in, 
               
               
                   
                   
                   
                   
                 respectively, 
               
               
                   
                   
                   
                   
                 Phillips et al., 
               
               
                   
                   
                   
                   
                 1998; Savkur et 
               
               
                   
                   
                   
                   
                 al.; Carlet et al., 
               
               
                   
                   
                   
                   
                 2002 &amp; Mankodi 
               
               
                   
                   
                   
                   
                 et al., 2002 
               
               
                   
               
               
                 Fibronectin 
                 ED I 
                 TGF-B, injury to 
                 GAAGAAGAC 
                 Kornblihtt et al., 
               
               
                   
                   
                 epithelial cells 
                   
                 1996 and ref 
               
               
                   
                   
                   
                   
                 therein 
               
               
                   
               
               
                 Beta- 
                 6A and 6B 
                 6A in non-muscle 
                 G-rich enhancer 
                 Gallego et al., 
               
               
                 tropomyosin 
                   
                 and smooth 
                   
                 1996 and ref 
               
               
                   
                   
                 muscle; 6B in 
                   
                 therein; Gooding 
               
               
                   
                   
                 skeletal muscle 
                   
                 et al., 2008 and 
               
               
                   
                   
                   
                   
                 ref therein 
               
               
                   
               
               
                 Enah 
                 11a 
                 Epithelial 
                 Fox2 and ESRP 
                 Warzecha et al, 
               
               
                   
                   
                   
                 sites 
                 2009a and 
               
               
                   
                   
                   
                   
                 2009b, and ref 
               
               
                   
                   
                   
                   
                 therein 
               
               
                   
               
               
                 Caspase 2, Slo 
                 Caspase 2 exon 9, Slo K+ 
                 Central nervous 
                 Caspase 2: 
                 Barash et al., 
               
               
                 K+ channel 
                 channel STREX exon, 
                 system 
                 secondary 
                 2010 
               
               
                   
                   
                   
                 structure region, 
                   
               
               
                   
                   
                   
                 Nova sites, 
                   
               
               
                   
                   
                   
                 (n)PTB sites; Slo 
                   
               
               
                   
                   
                   
                 K+ channel: 
                   
               
               
                   
                   
                   
                 Nova sites, 
                   
               
               
                   
                   
                   
                 (n)PTB sites 
                   
               
               
                   
               
               
                 PARD3, PTBP1, 
                 Exons were not named, 
                 Epithelial 
                 Fox2 sites 
                 Yeo et al., 2009 
               
               
                 ENAH 
                 primers flanking are 
                   
                   
                 supplemental 
               
               
                   
                 given: PARD3  
                   
                   
                 data 
               
               
                   
                 cassette flanked by 
                   
                   
                   
               
               
                   
                 CCAGTTCTTGCTTTTCAACGA 
                   
                   
                   
               
               
                   
                 and 
                   
                   
                   
               
               
                   
                 TCCCCATTCAAAGTCACCTC, 
                   
                   
                   
               
               
                   
                 PTBP1 cassette flanked by 
                   
                   
                   
               
               
                   
                 AGAACATCTACAACGCCTGC 
                   
                   
                   
               
               
                   
                 and 
                   
                   
                   
               
               
                   
                 TCTGGGTTGAGGTTGCTGAC 
                   
                   
                   
               
               
                   
                 ENAH cassette flanked by: 
                   
                   
                   
               
               
                   
                 TGCTTCAGCCTGTCATAGTCA 
                   
                   
                   
               
               
                   
                 and 
                   
                   
                   
               
               
                   
                 TGGCAGCAAGTCACCTGTTA 
               
               
                   
               
            
           
         
       
     
     Nonlimiting Exemplary Cell-Specific RNA Stability Elements 
     Various cell-specific RNA stability elements are known in the art. In some embodiments, a cell-specific RNA stability element is a microRNA target site. Many cell-specific microRNAs are known in the art. Nonlimiting exemplary mammalian cell-specific microRNAs are shown in Table 3. 
     In some embodiments, a cell-specific microRNA is a plant microRNA. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Nonlimiting exemplary cell-specific microRNAs 
               
            
           
           
               
               
               
               
            
               
                 microRNA 
                 Cell/tissue specificity  
                 microRNA 
                 Tissue specificity 
               
               
                   
               
               
                 miR-1a, d 
                 heart 
                 miR-122 
                 liver 
               
               
                 miR-124a, b 
                 brain 
                 miR-219 
                 brain 
               
               
                 Let-7c 
                 midbrain 
                 miR-154 
                 brain 
               
               
                 miR-375 
                 Pancreatic islets 
                 miR-125a, b 
                 brain 
               
               
                 miR-128a, b 
                 brain 
                 miR-127 
                 brain 
               
               
                 miR-10a, b 
                 Kidney 
                 miR-218 
                 Brain 
               
               
                 miR-30a-3p 
                 Kidney, lung, muscle 
                 miR-204 
                 Brain 
               
               
                 miR-148a 
                 liver 
                 miR-133a, b 
                 Heart, muscle 
               
               
                 miR-208 
                 Heart 
                 miR-215 
                 Intestine 
               
               
                 miR-194 
                 Intestine, kidney, liver 
                 miR-31 
                 Intestine, liver 
               
               
                 miR-141 
                 Intestine, kidney, lung 
                 miR-10a 
                 Intestine, kidney,  
               
               
                   
                   
                   
                 lung, spleen 
               
               
                 miR-150 
                 spleen 
                 miR-142-5p, 3p 
                 spleen 
               
               
                 miR-126 
                 Endothelial cells 
                 miR-155 
                 Hematopoietic cells 
               
               
                 miR-142 
                 Hematopoietic cells 
                 miR-181 
                 Hematopoietic cells 
               
               
                 miR-223 
                 Hematopoietic cells 
                 miR-140 
                 cartilage 
               
               
                 miR-206 
                 muscle 
               
               
                   
               
            
           
         
       
     
     Nonlimiting Exemplary Constructs 
     In some embodiments, a construct for synthetic regulation of gene expression is provided. In some such embodiments, the construct comprises a cell-specific promoter and a cell-specific exon. In some embodiments, the construct further comprises a cell-specific RNA stability element. 
     In some embodiments, a construct may further contain one or more additional elements that facilitate the propagation, use, and/or functioning of the construct, such as, without limitation, one or more coding sequences for selectable markers, one or more origins of replication, localization domains, etc. Elements for use in constructs for in vitro and in vivo gene expression are known in the art, and one skilled in the art can select suitable elements to include in a construct for synthetic regulation of gene expression described herein. In some embodiments, the selected elements facilitate the propagation, use, and/or functioning of a construct in a mammal. In some embodiments, the selected elements facilitate propagation, use, and/or functioning of a construct in a plant. In some embodiments, an element facilitates propagation of a construct in vitro, although the construct is intended for use in vivo. 
     Nonlimiting Exemplary Methods 
     In some embodiments, methods of synthetic regulation of gene expression in a cell, mammal, or plant are provided. In some embodiments, methods of cell-specific expression of a gene in a cell, mammal, or plant are provided. In some embodiments, a method comprises introducing into a cell, mammal, plant, or introducing into a selected cell in a mammal or plant, a construct comprising a cell-specific promoter, a heterologous gene, and a cell-specific exon, under conditions allowing expression of the heterologous gene in the selected cell. In some embodiments, a construct further comprises a cell-specific RNA stability element. 
     In some embodiments, the heterologous gene is expressed in the selected cell at levels at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is expressed in one or more other cells of the same organism. In some embodiments, the heterologous gene is expressed in the selected cell at levels at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is expressed in other cells of the organism. In some embodiments, the heterologous gene is expressed in a set of selected cells at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold greater than it is expressed in other cells of the organism. A set of selected cells may be any combination of cells in which a particular construct will express a heterologous gene. Nonlimiting exemplary sets of cells include, but are not limited to, cells of the cortex, hippocampus, and cerebellum; epithelial cells located in various tissues (such as kidneys and mammary glands); and muscle cells located throughout the body (such as skeletal muscle). 
     In some embodiments, a method comprises gene therapy in a mammal. In some such embodiments, the method allows expression of a heterologous gene in a selected cell type, with little or no expression of the heterologous gene in one or more other cell types. In some embodiments, a method comprises creating a transgenic animal. In some embodiments, a method comprises creating a transgenic plant. In some such embodiments, the method allows expression of a heterologous gene in a selected cell in the plant, with little or no expression of the heterologous gene in one or more other cells in the plant. In some embodiments, a method comprises creating transgenic fungi, for example, for temporal control of gene expression. 
     In some embodiments, the heterologous gene is expressed at a higher level in a selected cell and/or is expressed at a lower level in one or more other cells, than it would be expressed if it were only under the control of a cell-specific promoter. 
     The following examples are illustrative and are not intended to limit the claimed and/or disclosed subject matter. 
     EXAMPLES 
     Example 1: Synthetic Regulation of Gene Expression in Mesenchymal and Epithelial Cells 
     To demonstrate synthetic regulation of gene expression, a set of plasmids was designed that would provide differential expression of firefly luciferase in mesenchymal and epithelial cells. As shown in  FIG. 1A , four plasmids were designed. The control plasmid, pFFint, contains a firefly luciferase gene with a single intron that is spliced in both mesenchymal and epithelial cells, under the control of a CMV promoter, which is also active in both mesenchymal and epithelial cells. Plasmid pFFIIIc also contains a firefly luciferase gene under the control of a CMV promoter, but the gene contains the FGFR2 exon IIIc, which is efficiently spliced out in epithelial cells, but included in mesenchymal cells, resulting in an interruption of the luciferase open reading frame. Plasmid pE-Cad FFint contains a firefly luciferase gene with a single constitutive intron that is spliced in both mesenchymal and epithelial cells, but is under the control of the E-cadherin promoter, which is more active in epithelial cells than mesenchymal cells. Plasmid pE-cad-FFIIIc contains a firefly luciferase gene with the regulated FGFR2 exon IIIc, under the control of the E-cadherin promoter. 
     Mesenchymal-like rat prostate cancer cells, AT3 cells, and epithelial-like rat prostate cancer cells, DT cells, were grown (separately) in 6-well plates overnight in low glucose DMEM. DT and AT3 cells are described, for example, in Tennant et al. (2000). Each type of cell was transfected with 50 ng of each of the plasmids shown in  FIG. 1A , along with 10 ng of a constitutive renilla luciferase as a transfection control, and 1.95 μg pUC19 as carrier DNA. The cells were incubated at 37° C. overnight following transfection. The cells were then lysed and lysates were cleared by centrifuging for 10 minutes at 20,000 rcf (relative centrifugal force, or xg) at 4° C. Luciferase activities in the cell lysates were determined using the Dual Luciferase Assay System (Promega, Madison, Wis.). The firefly signal was divided by the control renilla signal for each plasmid/cell combination. 
     The results of that experiment are shown in  FIG. 1B . Similar firefly luciferase activities were observed for mesenchymal and epithelial cells transfected with control plasmid FFint. Firefly luciferase expression was higher in epithelial cells than in mesenchymal cells transfected with either plasmid FFIIIc (“FF3c” in  FIG. 1B ) or plasmid E-Cad FFint. The greatest expression difference was provided by plasmid E-Cad FFIIIc (“E-Cad FF3c” in  FIG. 1B ), which contains both an epithelial-specific splicing cis-acting elements and an epithelial-specific promoter. These results demonstrate that by providing two levels of control, such as a cell-specific promoter and a cell-specific intron, greater expression differentials between cell types can be obtained. 
     Example 2: Synthetic Regulation of Gene Expression in Mesenchymal and Epithelial Cells Using a Cre/Lox System 
     A system was designed to provide synthetic regulation of Cre expression in mesenchymal and epithelial cells, which would lead to a color change from red to green predominantly in epithelial cells.  FIG. 2A  shows the E-cadCreIIIc plasmid, which contains a Cre gene with the FGFR2 exon IIIc, under the control of the E-cadherin promoter. In mesenchymal cells, an inactive Cre fusion with FGFR2 exon IIIc will be expressed, which in epithelial cells, the FGFR2 exon IIIc will be spliced out and an active Cre protein will be expressed. The RG plasmid is also shown, which contains a dsRED gene flanked by loxP sites, followed by an EGFP gene. If an active Cre is produced in the cells, the dsRED gene will be removed, and the cells will express EGFP and be green. If an active Cre is not produced, the cells will express dsRED only and be red. The red circles in the RG plasmid indicated stop codons. 
     AT3 cells were transfected with 250 ng RG plasmid, 250 ng EcadCreIIIc plasmid, and 1.5 μg pUC19 as carrier DNA using lipofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer&#39;s instructions. Dt cells were transfected with 100 ng RG plasmid, 100 ng EcadCreIIIc plasmid, and 1.8 μg pUC19 as carrier DNA also using lipofectamine according to the manufacturer&#39;s instructions. The cells were incubated at 37° C. overnight. 
       FIG. 2B  is a picture of the red channel (left panels) and green channel (right panels) of transfected mesenchymal (top panels) and epithelial cells (bottom panels). Green cells were detected in the epithelial cell field, but not in the mesenchymal cell field. 
     The fraction of EGFP-positive cells was then tracked over time during selection for stable transfectants using hygromycin (selecting for the RG plasmid) and blastocidin (selecting for the EcadCreIIIc plasmid). Those results are shown in  FIG. 2C . At all time points except one, only epithelial cells expressed detectable EGFP. These results suggest that the combination of the E-cadherin promoter and FGFR2 exon IIIc effectively limited Cre expression to epithelial cells. 
     Example 3: Synthetic Regulation of Toxin Expression 
     To determine whether the synthetic regulation systems discussed herein can be used to limit toxin expression to particular cell types, a plasmid was designed to express diphtheria toxin only in epithelial cells.  FIG. 3A  shows plasmid EcadDipIIIc, which contains a diphtheria toxin gene with an FGFR2 exon IIIc within the coding region, under the control of the E cadherin promoter. In mesenchymal cells, little transcript will be expressed, and the transcript that is expressed should retain the FGFR2 IIIc exon, resulting in an inactive protein product. In epithelial cells, the transcript is more strongly expressed, and the FGFR2 exon IIIc is skipped, resulting in active diphtheria toxin protein expression and cell death. 
     AT3 cells were transfected with 200 or 2,000 ng CMV-DipIIIc plasmid and 1.8 or 0 μg pUC19 as carrier DNA using lipofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer&#39;s instructions. DT cells were transfected with 20 or 100 ng CMV-DipIIIc plasmid and 1.95 or 1.9 μg pUC19 as carrier DNA also using lipofectamine according to the manufacturer&#39;s instructions. The cells were incubated at 37° C. overnight with blasticidin selection, which selects for the CMV-DipIIIc plasmid. The presence or absence of the FGFR2 IIIc exon flanked by diphtheria toxin coding sequences was detected in mRNA isolated from the transfected cells using RT-PCR. 
     The results of that experiment are shown in  FIG. 3B . In mesenchymal cells, exon IIIc was detected using diphtheria-specific primers, which little or no exon IIIc was detected in transcripts from epithelial cells, as would be expected. Little or no transcript lacking exon IIIc (and therefore encoding active diphtheria toxin) was detected in epithelial cells, suggesting that the cells that expressed the correctly spliced diphtheria toxin were killed. While not intending to be bound by any particular theory, the surviving epithelial cells may have eliminated the diphtheria toxin-expressing plasmid, may not express the transcript at all, and/or may express a defective diphtheria toxin protein. It should also be noted that transfection efficiency could not be controlled in this experiment. 
     Example 4: Mesenchymal-Specific Synthetic Regulation of Expression 
     A mesenchymal-specific expression vector was created using a vimentin promoter and by including FGFR2 exons IIIb and IIIc. Exon IIIb is skipped in mesenchymal cells, while exon IIIc is retained. A diagram of the vector is shown in  FIG. 4A . The vector is shown in the center, with the various splice products shown above and below and labeled i, ii, iii, and iv. Splice product i results from excision of both exons IIIb and IIIc, and causes a frameshift in exemplary open reading frame (ORF) x and an inactive product. Splice product ii results from inclusion of exon IIIb and excision of exon IIIc (e.g., in epithelial cells), and results in a transcript with a stop codon ahead of ORF x. Splice product iii results from inclusion of both exons IIIb and IIIc, and results in a stop codon ahead of ORF x. Splice product iv results from exclusion of exon IIIb and inclusion of exon IIIc, resulting in a fusion of exon IIIc and ORF x in the correct reading frame and an active x fusion protein. Nonlimiting exemplary ORF x include luciferase, GFP, RFP, Cre, and DipA. 
     AT3 cells grown for one day in 6-well plates were transfected with 250 ng VimIIIbIIIc plasmid and 1.75 μg pUC19 as carrier DNA using lipofectamine (Invitrogen, Carlsbad, Calif.) according to the manufacturer&#39;s instructions. Dt cells were transfected with 250 ng VimIIIbIIIc plasmid and 1.75 μg pUC19 as carrier DNA also using lipofectamine according to the manufacturer&#39;s instructions. The cells were incubated at 37° C. overnight in low glucose DMEM. RNA was extracted from the cells using RNeasy (Qiagen) and the various splice products amplified by RT-PCR as previously described. See, e.g., Baraniak et al.,  Mol. Cell Biol.  2006 February; 26(4):1209-22. The amplified products were then cleaved using AvaI or HincII, which cleave the four splice products in such a way that products of various sizes can be used to identify each of the four splice products. Uncut RT-PCT products separate into a longer band, which corresponds to inclusion of both exons IIIb and IIIc (“double inclusion”), and a shorter band, which corresponds to inclusion of either exon IIIb or IIIc (“single inclusion”). AvaI cleaves once in the IIIb exon. HincII cleaves twice in the IIIc exon. See, e.g., Carstens et al.,  Mol. Cell Biol.,  20(19): 7388-7400 (2000), which is incorporated by reference herein in its entirety for any purpose. 
     The results of that experiment are shown in  FIG. 4B . The mesenchymal AT3 cells produced splice product iv, which includes exon IIIc only, whereas the epithelial DT cells did not produce appreciable levels of that splice product. Those results confirm that the VimxIIIbIIIc plasmid produces splice product iv, and presumably an active x fusion protein, in mesenchymal cells, but not epithelial cells. These results also demonstrate that changing the promoter from E-cadherin to vimentin does not affect the specificity of the splicing. 
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