WO2018187216A1

WO2018187216A1 - Expression cassettes and methods for detecting transcription errors

Info

Publication number: WO2018187216A1
Application number: PCT/US2018/025701
Authority: WO
Inventors: Jeffrey N. Strathern; Mary Kay ERNST; Alison Jean RATTRAY
Original assignee: The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2017-04-03
Filing date: 2018-04-02
Publication date: 2018-10-11

Abstract

Disclosed are collections of expression cassettes which may be useful for detecting transcription errors. The collection comprises (a) a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(n)G, wherein n is an integer from 7 to 20 and n is selected to place the DNA sequence of (i) out of the open reading frame by the absence of one nucleotide; and (b) a second expression cassette comprising a DNA sequence encoding a reporter gene and comprising a spacer sequence, wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase. Methods of (i) detecting transcription errors and (ii) screening test agents for the ability to increase transcription errors are also disclosed.

Description

EXPRESSION CASSETTES AND METHODS FOR DETECTING TRANSCRIPTION

ERRORS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 62/480,747, filed April 3, 2017, which is incorporated by reference in its entirety herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] This invention was made with Government support under project number ZIABC010992 by the National Institutes of Health, National Cancer Institute. The

Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Living cells transcribe DNA into messenger RNA (mRNA) by a process referred to as "transcription." RNAs can also have structural and regulatory roles. The cells translate the mRNA into proteins which contribute to the functioning of the living cell. Errors in transcription may lead to the synthesis of defective proteins which may, in turn, impair the health of the living cell. Transcription errors may contribute to diseases, e.g., aging-related diseases. Despite advancements in molecular biology, obstacles to the detection of transcription errors remain. Accordingly, there is a need for improved compositions and methods for the detection of transcription errors.

BRIEF SUMMARY OF THE INVENTION

[0004] An embodiment of the invention provides a collection of expression cassettes, the collection comprising: (a) a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(_n)G, wherein n is an integer from 7 to 20 and n is selected to place the DNA sequence of (i) out of the open reading frame by the absence of one nucleotide, and wherein (ii) is upstream of (i); and (b) a second expression cassette comprising a DNA sequence encoding a reporter gene and comprising a spacer sequence, wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase; wherein an error in transcription of (ii) of the first expression cassette produces a Cre recombinase transcript in the open reading frame such that active Cre recombinase is expressed and the expressed active Cre recombinase catalyzes removal of the spacer sequence in the second expression cassette so that the reporter gene is expressed; and wherein a lack of an error in transcription of (ii) of the first expression cassette produces a transcript out of the open reading frame for the Cre recombinase such that no active Cre recombinase is expressed, no active Cre recombinase catalyzes removal of the spacer sequence in the second expression cassette, and the reporter gene is not expressed.

[0005] Another embodiment of the invention provides a method of detecting a transcription error, the method comprising: transcribing a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(_n)G, wherein n is an integer from 7 to 20 and n is selected to place the DNA sequence of (i) out of the open reading frame by the absence of one nucleotide, and wherein (ii) is upstream of (i); providing a second expression cassette comprising a DNA sequence encoding a reporter gene and comprising a spacer sequence, wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase; wherein transcribing the first expression cassette comprises making (a) an error or (b) no error in transcription of (ii) of the first expression cassette, wherein making an error in transcription of (ii) of the first expression cassette produces a Cre recombinase transcript in the open reading frame, and the method further comprises expressing active Cre recombinase from the Cre recombinase transcript, catalyzing removal of the spacer sequence in the second expression cassette by the active Cre recombinase, and expressing the reporter gene; wherein making no error in transcription of (ii) of the first expression cassette produces a transcript out of the open reading frame for the Cre recombinase, wherein the method further comprises expressing no active Cre recombinase, not catalyzing removal of the spacer sequence in the second expression cassette, and not expressing the reporter gene; detecting expression or lack of expression of the reporter gene; wherein expression of the reporter gene is indicative of a transcription error and a lack of expression of the reporter gene is indicative of a lack of a transcription error. [0006] Another embodiment of the invention provides a method of screening a test agent for the ability to increase or decrease transcription errors. The method comprises providing test cells, wherein the test cells comprise: (a) a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(_n)G, wherein n is an integer from 7 to 20 and n is selected to place the DNA sequence of (i) out of the open reading frame by the absence of one nucleotide, and wherein (ii) is upstream of (i); and (b) a second expression cassette comprising a DNA sequence encoding a reporter gene and comprising a spacer sequence, wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase. The method further comprises: treating the test cells with a test agent and transcribing the first expression cassette in the treated test cells; wherein transcribing (ii) of the first expression cassette comprises making (a) an error or (b) no error in transcription of (ii) of the first expression cassette, wherein making an error in transcription of (ii) of the first expression cassette produces a Cre recombinase transcript in the open reading frame, and the method further comprises expressing active Cre recombinase from the Cre recombinase transcript, catalyzing removal of the spacer sequence in the second expression cassette by the active Cre recombinase, and expressing the reporter gene; wherein making no error in transcription of (ii) of the first expression cassette produces a transcript out of the open reading frame for the Cre recombinase, wherein the method further comprises expressing no active Cre recombinase, not catalyzing removal of the spacer sequence in the second expression cassette, and not expressing the reporter gene. The method further comprises: providing control cells, wherein the control cells are identical to the test cells except that the control cells are not treated with the test agent; measuring expression of the reporter gene in the control cells and in the test cells; and comparing the expression of the reporter gene in the control cells to the expression of the reporter gene in the test cells; wherein an increase in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent increases transcription errors; wherein a lack of an increase in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent does not increase transcription errors; wherein a decrease in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent decreases transcription errors; and wherein a lack of a decrease in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent does not decrease transcription errors. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] Figure 1 A is a schematic illustrating an example of a first expression cassette according to the invention. The first expression cassette comprises an open reading frame (ORF) and comprising (i) a DNA sequence encoding Cre recombinase ("ere") and (ii) a DNA sequence encoding A(io)G, wherein (ii) is upstream of (i). ATG=translation start.

MB=missing base. SA=splice acceptor. In Figure 1A, A(_n)G is A(io)G.

[0008] Figure IB is a schematic illustrating an example of a second expression cassette comprising a DNA sequence encoding a reporter gene (enhanced yellow fluorescent protein (eYFP)) and comprising a spacer sequence (STOP), wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase.

[0009] Figure 1C is a schematic illustrating the second expression cassette of Figure IB following removal of the spacer sequence (STOP) by Cre recombinase.

[0010] Figure 2 is a schematic illustrating an example of misincorporation-induced slippage.

DETAILED DESCRIPTION OF THE INVENTION

[0011] The inventive collections of expression cassettes, methods of detecting a transcription error, and methods of screening a test agent for the ability to increase transcription errors may provide any of a variety of advantages. For example, prior to the invention, mRNA transcription errors were difficult or impossible to detect due to any one or more of (i) the short half-life of the mRNA molecule, (ii) the instability of the mRNA molecule, (iii) the high error rate of protein translation, and (iv) the lack of a stable phenotypic change that results from the transcription error. By way of illustration, errors in DNA synthesis result in permanent mutations which are easy to detect. In contrast, mRNA transcription errors result in no stable phenotypic change and were difficult or impossible to detect prior to the invention. The inventive compositions and methods advantageously cause transcription errors to result in a permanent genetic change that may be observed as a stable phenotypic change, thereby overcoming these obstacles to the detection of transcription errors. Moreover, transcription errors may be rare events which may occur, for example, in one out of a million transcripts or in a small fraction of cells, e.g., one out of a thousand cells. The inventive compositions and methods advantageously make it possible to detect such rare transcription errors.

[0012] Without being bound to a particular theory or mechanism, it is believed that transcription errors may contribute to aging, age-related diseases, and the formation of misfolded proteins. Accordingly, the inventive compositions and methods are contemplated to be useful for any of a variety of applications including, but not limited to, (i) the study of the role of transcription errors in human health and disease (e.g., overall health, fertility, aging, dementia, and organ toxicity); (ii) the screening for agents which may increase or decrease transcription errors; (iii) the study of the genetics of transcription fidelity; (iv) the study of factors involved in reversing misincorporation (e.g., by inducing the RNA polymerase to cleave off the misincorporated base at the 3' end of the RNA); and (v) the study of the transcription fidelity of RNA polymerase mutants in tumors.

[0013] An embodiment of the invention provides a collection of expression cassettes. The collection comprises a first expression cassette. An example of a first expression cassette is shown in Figure 1A. The first expression cassette comprises an open reading frame (ORF) and a DNA sequence encoding Cre recombinase. The first expression cassette further comprises a DNA sequence encoding A(_n)G, wherein n is an integer from 7 to 20. With respect to A(_n)G, "n" may be any integer from 7 and 20, for example, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or a range defined by any of the foregoing values. In an embodiment of the invention, the "n" of A(_n)G is an integer from 8 to 20, from 8 to 15, or from 8 to 10. The "n" of A(_n)G is also selected to place the DNA sequence encoding Cre recombinase out of the ORF by the absence of one nucleotide. As shown in Figure 1A, the DNA sequence encoding A(_n)G is upstream of the DNA sequence encoding Cre recombinase. In an embodiment of the invention, the first expression cassette further comprises a selectable gene.

[0014] The inventive collection of expression cassettes further comprises a second expression cassette. An example of a second expression cassette is shown in Figure IB. The second expression cassette comprises a DNA sequence encoding a reporter gene and comprising a spacer sequence. The spacer sequence is flanked by loxP sites. The spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase.

[0015] The reporter gene may be any reporter gene known in the art. Suitable reporter genes may include, but are not limited to, any of fluorescent protein (e.g., green (GFP), red, yellow, or cyan fluorescent protein, enhanced green, red, yellow, or cyan fluorescent protein), beta-lactamase, beta-galactosidase, luciferase (e.g., firefly luciferase (FLuc), Renilla (RLuc) luciferase, NANOLUC luciferase (NlucP) (Promega, Madison, WI), bacterial luciferase, Click-Beetle Luciferase Red (CBRluc), Click-Beetle Luciferase Green (CBG681uc and CBG991uc), Metridia pacifica Luciferase (MetLuc), Gaussia Luciferase (GLuc), Cypridina Luciferase, and Gaussia-Dura Luciferase), chloramphenicol acetyltransferase (CAT), neomycin phosphotransferase, alkaline phosphatase, secreted alkaline phosphatase (SEAP), mCherry, tdTomato, TurboGFP, TurboRFP, dsRed, dsRed2, dsRed Express, AcGFPl, ZsGreenl, Red Firefly Luciferase, Enhanced Click-Beetle Luciferase (ELuc), Dinoflagellate Luciferase, Pyrophorus plagiophthalamus Luciferase (lucGR), Bacterial luciferase (Lux), pmeLUC, Phrixothrix hirtus Luciferase, Gaussia-Dura Luciferase, RenSP, Vargula hilgendorfii Luciferase, Lucia Luciferase, Metridia longa Luciferase (MetLuc), HaloTag, SNAP-tag, CLIP-tag, β-Glucuronidase, Aequorin, Secreted placental alkaline phosphatase (SPAP), Gemini, TagBFP, mTagBFP2, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T- Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, mTurquoise2, Midoriishi-Cyan, TagCFP, mTFPl, Emerald, Superfolder GFP, Azami Green, TagGFP2, mUKG, mWasabi, Clover, Citrine, Venus, SYFP2, TagYFP, Kusabira-Orange, mKO, mK02, mOrange, mOrange2, mRaspberry, mStrawberry, mTangerine, TagRFP, TagRFP-T, mApple, mRuby, mRuby2, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKatel, LSS-mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGRl (green), KikGRl (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), mEos3.2 (green), mEos3.2 (red), PSmOrange, PSmOrange, Dronpa, TurboYFP, TurboFP602 , TurboFP635, TurboFP650, hrGFP, hrGFP II, E2-Crimson, HcRedl, Dendra2, AmCyanl, ZsYellowl, mBanana , EBFP, Topaz, mECFP, CyPet, yPet, PhiYFP, DsRed-Monomer, Kusabira Orange, Kusabira Orange2, Jred, AsRed2, dKeima-Tandem, AQ143, mKikGR, HIS3, ADE2, MET15, and ADE6.

[0016] The spacer sequence is not limited and may include any DNA sequence which prevents expression of the reporter gene in the absence of Cre recombinase. Suitable spacer sequences may include, but are not limited to, one or both of any selectable gene (such as, for example, a drug resistance gene) and a transcription terminator to block transcription of the reporter gene in the absence of Cre recombinase. When a population of cells is modified (e.g., transduced or transfected) with the expression cassette containing the spacer sequence, some cells of the population may successfully take the expression cassette up and other cells of the population may not. A selectable gene (such as, for example, a drug resistance gene) may be useful for selecting only those cells which have been successfully modified to comprise the spacer sequence including the drug resistance gene. For example, a population of cells may be modified to contain a spacer sequence with a drug resistance gene, and then the population of cells may be initially plated in medium containing the drug. Those cells which do not contain the spacer sequence with the drug resistance gene will die, leaving only those living cells containing the spacer sequence including the drug resistance gene on the plate. Those living cells may then be re-plated. Accordingly, a spacer sequence including a selectable gene may, advantageously, reduce background when measuring reporter gene expression. In an embodiment of the invention, the first and/or second expression cassettes further comprise a promoter and/or a splice acceptor.

[0017] The collection of expression cassettes may be stably transfected into the cells. In this regard, the collection of expression cassettes may become integrated into the respective genomes of the transfected cells and may, therefore, be replicated. Descendents of the transfected cells also include the collection of expression cassettes, resulting in stably transfected cells. In this regard, the inventive compositions and methods advantageously cause transcription errors to result in a permanent genetic change that may be observed as a stable phenotypic change.

[0018] An error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette produces a Cre recombinase transcript in the ORF for Cre recombinase such that active Cre recombinase is expressed. The expressed, active Cre recombinase catalyzes removal of the spacer sequence in the second expression cassette so that the reporter gene is expressed (e.g., stably (that is, permanently) expressed). An example of a Cre recombinase-catalyzed second expression cassette is shown in Figure 1C. As shown in Figure 1C, the spacer sequence (STOP) has been removed by Cre recombinase so that the reporter gene (YFP) may be expressed. Expression of the reporter gene indicates that a transcription error of the DNA sequence encoding A(_n)G of the first expression cassette occurred.

[0019] The lack of an error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette produces a transcript in the wrong reading frame for the Cre recombinase such that no active Cre recombinase is expressed. In other words, the lack of an error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette produces a transcript which is out of the ORF for the Cre recombinase such that no active Cre recombinase is expressed. The lack of an error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette produces an out-of-frame Cre recombinase transcript. In this regard, no active Cre recombinase catalyzes removal of the spacer sequence in the second expression cassette. Consequently, the reporter gene is not expressed. The lack of reporter gene expression indicates that no transcription error of the DNA sequence encoding A(_n)G of the first expression cassette occurred.

[0020] In an embodiment of the invention, the error in transcription includes a misincorporation of a nucleotide into a transcript of the DNA sequence encoding A(_n)G which the DNA sequence encoding A(_n)G does not encode. A misincorporation transcription error involves the substitution of an incorrect nucleotide into the transcript which is not encoded by the corresponding position in the template DNA molecule. For example, error-free transcription of DNA including TGT results in a transcript including UGU, while a misincorporation error of the same DNA results in UAU.

[0021] In an embodiment of the invention, the error in transcription is (a) addition of a nucleotide into a transcript of the DNA sequence encoding A(_n)G which the DNA sequence encoding A_(n)G does not encode or (b) deletion of two nucleotides from a transcript of the DNA sequence encoding A(_n)G which are encoded in the DNA sequence encoding A(_n)G. In this regard, the inventive collection of expression cassettes may detect "slippage" transcription errors. A "slippage" transcription error constitutes a temporary loss of DNA register by NA polymerase leading to one or both of insertions and deletions of nucleotides in mRNA. For example, RNA polymerase may make one or both of insertions and deletions of nucleotides when the DNA template has a repeat of the same base that is longer than the length of the RNA/DNA hybrid inside the RNA polymerase enzyme. For example, a DNA template with a homopolymeric tract of 1 1 thymines (T) may be transcribed into a transcript having a homopolymeric tract of 12 adenines (A). A "slippage" transcription error may or may not be induced by a misincorporation error, as explained in more detail below. A "slippage" transcription error which is not induced by a misincorporation error is also referred to as a "non-misincorporation-induced slippage transcription error."

[0022] A "slippage" transcription error may be induced by a misincorporation error (also referred to as a "misincorporation-induced slippage transcription error"). For example, as shown in Figure 2, misincorporation of an adenine (A) in place of a guanine (G) may extend the length of the nascent transcript, which can readily translocate backwards because of the nature of the preceding polyA-tract. This leaves the cytosine (C) unpaired again, and gives the polymerase another opportunity to incorporate a G opposite of the C.

[0023] The inventive compositions and methods advantageously detect both

misincorporation-induced slippage transcription errors and non-misincorporation-induced slippage transcription errors. In the first expression cassette, the DNA sequence encoding A(n)G is out of frame with the DNA sequence encoding Cre recombinase because it is missing a nucleotide. A nucleotide needs to be added to the RNA transcript (or two nucleotides need to be deleted) in order to make active Cre recombinase. The necessary nucleotide can be added if (i) "slippage" of the transcript occurs, which adds a nucleotide to the transcript which is not encoded by the DNA (or deletes two nucleotides which are encoded by the DNA) in the absence of misincorporation (a non-misincorporation-induced slippage transcription error), or (ii) "misincorporation-induced slippage" occurs, which incorporates the incorrect nucleotide which is not encoded by the DNA and also adds a nucleotide which is not encoded by the DNA due to backward slippage of the transcript ("misincorporation- induced slippage transcription error"). Accordingly, the inventive compositions and methods advantageously detect both types of transcription "slippage" errors, thereby increasing the efficiency of the detection of transcription "slippage" errors.

[0024] Another embodiment of the invention provides one or more recombinant expression vectors comprising the inventive collection of expression cassettes. In this regard, an embodiment of the invention provides a first recombinant expression vector comprising any of the first expression cassettes of the invention described herein and a second recombinant expression vector comprising any of the second expression cassettes of the invention described herein. In another embodiment of the invention, a single recombinant expression vector comprises both the first and second expression cassettes of the invention.

[0025] For purposes herein, the term "recombinant expression vector" means a genetically-modified oligonucleotide or polynucleotide construct that permits the expression of an mRNA, protein, polypeptide, or peptide by a host cell, when the construct comprises a nucleotide sequence encoding the mRNA, protein, polypeptide, or peptide, and the vector is contacted with the cell under conditions sufficient to have the mRNA, protein, polypeptide, or peptide expressed within the cell. The vectors of the invention are not naturally-occurring as a whole. However, parts of the vectors can be naturally-occurring.

[0026] In an embodiment of the invention, the one or more recombinant expression vectors of the invention can include any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, LaJolla, CA), the pET series (Novagen, Madison, WI), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA). Bacteriophage vectors, such as λϋΤΙΟ, λΰΤΙ 1, λZapII

(Stratagene), EMBL4, and λΝΜΙ 149, also can be used. Examples of plant expression vectors include pBIOl, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, e.g., a retroviral vector.

[0027] In an embodiment of the invention, the one or more recombinant expression vectors of the invention can be prepared using standard recombinant DNA techniques described in, for example, Green et al. (eds.), Molecular Cloning, A Laboratory Manual, 4^th Edition, Cold Spring Harbor Laboratory Press, New York (2012). Constructs of expression vectors, which are circular or linear, can be prepared to contain a replication system functional in a prokaryotic or eukaryotic host cell. Replication systems can be derived, e.g., from ColEl, 2 μ plasmid, λ, SV40, bovine papilloma virus, and the like.

[0028] The recombinant expression vector may comprise additional regulatory sequences such as transcription and translation initiation and termination codons, which are specific to the type of host cell (e.g., bacterium, fungus, plant, or animal) into which the vector is to be introduced, as appropriate, and taking into consideration whether the vector is DNA- or RNA-based.

[0029] Another embodiment of the invention provides an isolated host cell comprising the inventive collection of expression cassettes or the inventive one or more recombinant expression vectors described herein. As used herein, the term "host cell" refers to any type of cell that can contain the inventive collection of expression cassettes or the inventive recombinant expression vector. For purposes of amplifying or replicating the recombinant expression vector, the host cell may be a prokaryotic cell, e.g., a DH5a cell. For purposes of providing a cell-based assay, the host cell can be a eukaryotic cell, e.g., plant, animal, yeast, fungi, or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa. The host cell can be a cultured cell or a primary cell, i.e., isolated directly from an organism, e.g., a human or a mouse. Preferably, the host cell is a mouse cell, a yeast cell, a bacterial cell, or a human cell. In an especially preferred embodiment, the host cell is a human cell. In an embodiment, the host cell is a mammalian cell. The host cell may be any type of mammalian cell including, but not limited to, a T cell, a B cell, a macrophage, a neutrophil, an erythrocyte, a hepatocyte, an endothelial cell, an epithelial cell, a muscle cell, or a brain cell, etc.

[0030] Also provided by an embodiment of the invention is a population of cells comprising at least one host cell described herein. The population of cells can be a heterogeneous population comprising the host cell comprising any of the inventive collections of expression cassettes or recombinant expression vectors described, in addition to at least one other cell, e.g., a cell which does not comprise any of the collections of expression cassettes or recombinant expression vectors. Alternatively, the population of cells can be a substantially homogeneous population, in which the population comprises mainly host cells (e.g., consisting essentially of) comprising the inventive collections of expression cassettes or recombinant expression vectors described herein. The population also can be a clonal population of cells, in which all cells of the population are clones of a single host cell comprising the collection of expression cassettes or recombinant expression vector, such that all cells of the population comprise the collection or expression cassettes or recombinant expression vector. In one embodiment of the invention, the population of cells is a clonal population comprising host cells comprising the collection of expression cassettes or recombinant expression vector as described herein.

[0031] The collections of expression cassettes, recombinant expression vectors, and host cells (including populations thereof) can be isolated and/or purified. The term "isolated" as used herein means having been removed from its natural environment. The term "purified" or "isolated" does not require absolute purity or isolation; rather, it is intended as a relative term. Thus, for example, a purified (or isolated) host cell preparation is one in which the host cell is more pure than cells in their natural environment within the body. Such host cells may be produced, for example, by standard purification techniques. In some embodiments, a preparation of a host cell is purified such that the host cell represents at least about 50%, for example at least about 70%, of the total cell content of the preparation. For example, the purity can be at least about 50%, can be greater than about 60%, about 70% or about 80%, or can be about 100%.

[0032] It is contemplated that the inventive collections of expression cassettes may be useful for studying transcription errors in tissues in vitro. In this regard, another embodiment of the invention provides an isolated tissue comprising any of the inventive collections of expression cassettes, recombinant expression vectors, host cells, or populations of host cells described herein. For purposes of providing a tissue-based assay, the tissue can be isolated from a plant or animal. In an embodiment, the tissue is a mammalian tissue. Preferably, the tissue is a mouse tissue or a human tissue. In an especially preferred embodiment, the tissue is a human tissue. The tissue may be any type of mammalian tissue including, but not limited to, blood, hepatic tissue, endothelial tissue, epithelial tissue, muscle tissue, liver tissue, thymic tissue, or brain tissue, etc.

[0033] It is contemplated that the inventive collections of expression cassettes may be useful for studying transcription errors in organs in vitro. In this regard, another embodiment of the invention provides an isolated organ comprising any of the inventive collections of expression cassettes, recombinant expression vectors, host cells, or populations of host cells described herein. For purposes of providing an organ-based assay, the organ can be isolated from an animal or may be grown in vitro. The organ may be a whole organ or a partial organ. In an embodiment, the organ is a mammalian organ. Preferably, the organ is a mouse organ or a human organ. The tissue may be any type of mammalian organ including, but not limited to, liver, bladder, heart, pancreas, etc.

[0034] It is contemplated that the inventive collections of expression cassettes may be useful for studying transcription errors in non-human animals in vivo. In this regard, another embodiment of the invention provides a non-human animal comprising any of the inventive collections of expression cassettes, recombinant expression vectors, host cells, or populations of host cells described herein. Preferably, the non-human animal is a non-human mammal. Examples of non-human mammals may include mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits. The non-human mammals may be from the order Carnivora, including Felines (cats) and Canines (dogs). The non-human mammals may be from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). The non-human mammals may be of the order Primates, Ceboids, or Simoids (monkeys). The inventive mouse may be crossed to any of a variety of mouse models of cancer, e.g., to determine the effect of different cancer types on transcription fidelity and/or to screen for treatments that could reverse any adverse effects of transcription errors.

[0035] It is contemplated that the inventive collections of expression cassettes, recombinant expression vectors, and host cells (including populations thereof) may be useful for carrying out methods of detecting transcription errors. In this regard, an embodiment of the invention provides a method of detecting a transcription error. The method comprises transcribing a first expression cassette. The first expression cassette is as described herein with respect to other aspects of the invention.

[0036] The method further comprises providing a second expression cassette. The second expression cassette is as described herein with respect to other aspects of the invention.

[0037] Transcribing the first expression cassette comprises making (a) an error or (b) no error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette.

[0038] Making an error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette produces a Cre recombinase transcript in the ORF (that is, an in-frame Cre recombinase transcript). When an error in transcription of the DNA sequence encoding A(n)G is made, the method further comprises expressing active Cre recombinase from the Cre recombinase transcript, catalyzing removal of the spacer sequence in the second expression cassette by the active Cre recombinase, and expressing the reporter gene.

[0039] Making no error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette produces a Cre recombinase transcript in the wrong ORF. In other words, making no error in transcription of the DNA sequence encoding A(_n)G of the first expression cassette produces a transcript out of the ORF for the Cre recombinase (that is, an out-of- frame Cre recombinase transcript). When no error in transcription of the DNA sequence encoding A(„)G is made, the method further comprises expressing no active Cre recombinase, not catalyzing removal of the spacer sequence in the second expression cassette, and not expressing the reporter gene.

[0040] The method further comprises detecting expression or lack of expression of the reporter gene, wherein expression of the reporter gene is indicative of a transcription error and a lack of expression of the reporter gene is indicative of a lack of a transcription error. Detecting expression (or lack of expression) of the reporter gene may be carried out in any suitable manner. For example, detecting expression (or lack of expression) of the reporter gene may include contacting the cells containing the first and second expression cassettes with one or more detection reagents that react(s) with the reporter gene to provide a detectable indicator (e.g., fluorescence, luminescence, and color changes) of the expression (or lack of expression) of the reporter gene. The detectable indicator may, for example, be a visible indicator. In an embodiment of the invention in which the reporter gene chosen does not require a detection reagent in order to provide a detectable indicator of the expression (or lack of expression) of the reporter (e.g., any of the fluorescent proteins such as green, red, yellow, or cyan fluorescent protein), detecting expression (or lack of expression) of the reporter may be carried out without contacting the cells with a detection reagent.

[0041] It is contemplated that the inventive collections of expression cassettes, recombinant expression vectors, and host cells (including populations thereof) may be useful for carrying out methods of screening a test agent for the ability to increase or decrease transcription errors. The method may comprise providing test cells. The test cells may comprise any of the host cells described herein with respect to other aspects of the invention. The test cells comprise a first expression cassette and a second expression cassette, which are as described herein with respect to other aspects of the invention. In an embodiment of the invention, the test cells comprise any of the recombinant expression vectors described herein with respect to other aspects of the invention.

[0042] The method comprises treating the test cells with a test agent. The test agent is not limited and may be any agent suspected of increasing or decreasing transcription errors. For example, the test agent may be a drug, protein, enzyme, antibody, or a small molecule. An example of a test agent is manganese. The treating of the test cells may be carried out in any suitable manner, for example, by directly physically contacting the test cells with the test agent.

[0043] The method further comprises transcribing the first expression cassette in the treated test cells. The transcribing of the DNA sequence encoding A(_n)G of the first expression cassette comprises making (a) an error or (b) no error in transcription of the DNA sequence encoding A(„)G of the first expression cassette, as described herein with respect to other aspects of the invention.

[0044] The method further comprises providing control cells. The control cells are identical to the test cells except that the control cells are not treated with the test agent.

[0045] The method further comprises measuring expression of the reporter gene in the control cells and in the test cells. Measuring expression of the reporter gene may include detecting the expression of the reporter gene as described herein with respect to other aspects of the invention. Measuring expression of the reporter gene may include observing and/or measuring the quantity of any one or more of fluorescence, luminescence, absorbance, and color changes, as is appropriate for particular reporter gene chosen.

[0046] The method further comprises comparing the expression of the reporter gene in the control cells to the expression of the reporter gene in the test cells. An increase in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent increases transcription errors. A lack of an increase in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent does not increase transcription errors. A decrease in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent decreases transcription errors. A lack of a decrease in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent does not decrease transcription errors.

[0047] In an embodiment of the invention, with respect to any of the inventive methods described herein, making an error in transcription may comprise misincorporating a nucleotide into a transcript of the DNA sequence encoding A(_n)G which the DNA sequence encoding A(_n)G does not encode and adding a nucleotide into the transcript of the DNA sequence encoding A(_n)G which the DNA sequence encoding A(_n)G does not encode. Such a transcription error is also referred to as a "mincorporation-induced slippage transcription error" and may be as described herein with respect to other aspects of the invention.

Mis incorporation errors may facilitate slippage errors that place the Cre recombinase in the first expression cassette in-frame.

[0048] In an embodiment of the invention, with respect to any of the inventive methods described herein, making an error in transcription may comprise (a) adding a nucleotide into a transcript of the DNA sequence encoding A(_n)G which the DNA sequence encoding A(_n)G does not encode or (b) deleting two nucleotides from a transcript of the DNA sequence encoding A(_n)G which are encoded in the DNA sequence encoding A(_n)G. Such a transcription error may occur in the absence of a misincorporation transcription error. The adding of a nucleotide (or the deleting of nucleotides) which occurs in the absence of a misincorporation transcription error is also referred to as a "non-misincorporation-induced slippage transcription error" and may be as described herein with respect to other aspects of the invention.

[0049] As explained herein with respect to other aspects of the invention, expression of the reporter gene is indicative of a transcription error. In some cases, it may also be useful to determine whether or not a DNA mutation occurred in the collection of expression cassettes in the modified cells which stably express the reporter gene. Accordingly, with respect to any of the inventive methods described herein, the method may further comprise obtaining DNA from one or more cells which have been modified to comprise the inventive collection of expression cassettes and assaying the DNA to detect a DNA mutation in at least one copy of the DNA encoding the collection of expression cassettes. The DNA may be copied or amplified from the cells (e.g., via polymerase chain reaction (PCR) or other suitable technique).

[0050] The method may further comprise comparing the DNA sequence of the collection of expression cassettes from the one or more modified cells to the DNA sequence of the one or more vectors used to modify the cells and identifying any differences between the DNA sequence of the collection of expression cassettes from the one or more modified cells to the DNA sequence of the vector used to modify the cells. For example, the method may comprise assaying the DNA to detect a DNA mutation in at least one copy of the DNA sequence encoding A(_n)G in the one or more modified cells. The absence of a DNA mutation in the DNA sequence encoding A(_n)G in the one or more modified cells, together with expression of the reporter gene, indicates that expression of the reporter gene is due to an error in transcription of the DNA sequence encoding A(_n)G and not a mutation in the DNA sequence encoding A(_n)G. The presence of a DNA mutation in the DNA sequence encoding A(_n)G in the one or more modified cells, together with expression of the reporter gene, indicates that expression of the reporter gene may be due to an error in transcription of the DNA sequence encoding A(_n)G or a mutation in the DNA sequence encoding A(_n)G.

[0051] With respect to any of the inventive methods described herein, the method may comprise detecting expression or lack of expression of the reporter gene in any of the isolated cells, tissues, organs, or non-human animals described herein with respect to other aspects of the invention. In an embodiment of the invention, the method comprises detecting expression or lack of expression of the reporter gene in isolated mouse cells, isolated human cells, or in a mouse.

[0052] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope.

EXAMPLE 1

[0053] This example demonstrates the preparation of transgenic mice including (a) a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(_n)G, wherein n is 10 and n is selected to place the DNA sequence of (i) out of the ORF by the absence of one nucleotide, and wherein (ii) is upstream of (i); and (b) a second expression cassette comprising a DNA sequence encoding a reporter gene and comprising a spacer sequence, wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase.

[0054] Expression cassettes were prepared including a first expression cassette and a second expression cassette. The first expression cassette is shown in Figure 1A and comprised an ORF and (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(io)G. The DNA sequence encoding Cre recombinase was out of the ORF by the absence of one nucleotide. The DNA sequence encoding A(io)G was positioned upstream of the DNA sequence encoding Cre recombinase.

[0055] The second expression cassette is shown in Figure IB and comprised a DNA sequence encoding enhanced yellow fluorescent protein (eYFP) and comprised a spacer sequence. The spacer sequence was flanked by loxP sites. The spacer sequence was positioned to prevent expression of the reporter gene in the absence of active Cre recombinase.

[0056] Transgenic Black6 mice were prepared including the first and second expression cassettes. The increase of YFP was measured in the blood lymphocytes of 26 control mice (Rows 1-3 of Table 1) and in 25 mice with the first and second expression cassettes. It was found that the average level of expression in the control mice was about 1.9 X 10^"5. The average level of expression in the mice with the first and second constructs was about 5.6 X 10^"4. DNA corresponding to the first expression cassette (Al OG-Cre) was isolated from the YFP positive blood lymphocytes and sequenced. The results showed that the YFP positive blood lymphocytes retained the A(10)G sequence consistent with the interpretation that YFP expression was not the consequence of a DNA mutation permanently allowing Cre expression, but rather a transcription error resulting in transient production of Cre and activation of the YFP expression cassette.

[0057] The eYFP expression in the mice was monitored. An increase in eYFP expression of about three-fold was measured over about 200 days. YFP was also monitored by immunohistology of various tissues from 14 mice with the first and second expression cassettes (7 male and 7 female) and from 4 control mice (3 female and 1 male). There was a detectable but low level of YFP patches in most tissues of the mice with the first and second expression cassettes. The size of the patch provided a sense of how early the transcription error occurred. EXAMPLE 2

[0058] This example demonstrates that cells comprising the first and second expression cassettes of Example 1 detect manganese-induced transcription errors.

[0059] Cell lines were isolated from the mice of Example 1 with the first and second expression cassettes. The cell lines were stable. Exposure of the cells to elevated levels of manganese resulted in an increase in the number of YFP-expressing cells. This result was consistent with in vitro experiments which demonstrated that manganese can reduce the accuracy of transcription.

EXAMPLE 3

[0060] This example demonstrates the proportion of lymphocytes comprising the first and second expression cassettes of Example 1 which express the reporter gene.

[0061] Transgenic mice were prepared as described in Table 1. Blood was taken from the mice and the lymphocytes were sorted to count the cells expressing enhanced yellow fluorescent protein (eYFP) or not expressing eYFP. The results are shown in Table 1.

TABLE 1

[0062] The mice in Row 1 of Table 1 have Cre in-frame (Cre+) and, therefore, all of the constructs were in the form of Figure 1 C and were YFP+.

[0063] The mice in Row 2 of Table 1 have neither the cassette of Fig.1 A nor the cassette of Fig. IB (no cre, no eYFP). [0064] The mice in Row 3 of Table 1 have no Cre cassette at all (Cre-) but had the cassette of Fig. IB (eYFP construct).

[0065] The mice in Row 4 of Table 1 have both of the cassettes of Fig. 1A and Fig. IB.

[0066] The bottom row of Table 1 shows the results obtained in the mice with the first and second expression cassettes of Example 1 (AioG MB Cre(eYFP)Cos) and shows that an average of 0.3% of the cells express eYFP (presumed to reflect cells (or progeny of cells) that had a transcription error (average of 3.06 x 10^"3)). Row 3 of Table 1 indicates that control mice with the eYFP reporter and no Cre source (No Cre/(eYFP)Cos) showed that the background was only about 1 per 100,000 cells (average of 1.10 x 10^"5).

EXAMPLE 4

[0067] This example demonstrates that cells comprising the first and second expression cassettes of Example 1 which express the reporter gene can be detected in stomach tissue.

[0068] Transgenic mice were prepared as follows, with reference to Table 1 : 14 mice containing both cassettes (Table 1, Row 4), 4 mice with the eYFP cassette only (Table 1, Row 3), 1 mouse with neither cassette (Table 1, Row 2), and 1 mouse having a construct with Cre in-frame (Table 1 , Row 1).

[0069] The mice were sacrificed, and cross sections of the stomach were

immunologically stained for YFP and stained with DAPI. The stained cross sections were examined under a microscope to assess the presence of staining. Cells which stained positive for YFP expression were observed as brown cells. These brown, YFP=positive cells reflected cells that had a transcription error in the DNA sequence encoding A(io)G.

[0070] Because the activation of YFP expression by Cre is a permanent reflection of the transcription error, the cell with the error can grow into a sector of cells in the tissue. The number of cells in a sector reflects how many generations ago the error occurred. Sectors of brown cells were observed in the stomach tissue.

[0071] Control mice without the DNA sequence encoding A(io)G showed almost no YFP expression.

EXAMPLE 5

[0072] This example demonstrates that cells comprising the first and second expression cassettes of Example 1 which express the reporter gene can be detected in brain tissue. [0073] Cross sections of the brains of the mice of Example 4 were immunologically stained for YFP using an antibody against YFP and stained with DAPI. The stained cross sections were examined under a microscope to assess the presence of staining. Cells which stained positive for YFP expression were observed as brown cells.

[0074] About 3-10 % of the cells in the brain cross sections from each of the four mice with the first and second expression cassettes of Example 1 showed YFP expression. The positive control showed strong staining in all of the cells throughout the cross section. None of the cells in the negative control showed activation of YFP expression.

EXAMPLE 6

[0075] This example demonstrates that cells comprising the first and second expression cassettes of Example 1 which express the reporter gene can be detected in small intestine tissue.

[0076] Cross sections of the small intestines of the mice of Example 4 were

immunologically stained for YFP using an antibody against YFP and stained with DAPI. The stained cross sections were examined under a microscope to assess the presence of staining. Cells which stained positive for YFP expression were observed as brown cells.

[0077] A small fraction of the cells in the small intestine cross section from the mouse with the first and second expression cassettes of Example 1 showed YFP expression. A sector of cells showing YFP expression was also visible as a brown patch in the cross section from the mouse with the first and second expression cassettes of Example 1. The positive control showed strong staining in all of the cells throughout the cross section. None of the cells in the negative control showed activation of YFP expression.

EXAMPLE 7

[0078] This example demonstrates that cells comprising the first and second expression cassettes of Example 1 which express the reporter gene can be detected in liver tissue.

[0079] Cross sections of the livers of the mice of Example 4 were immunologically stained for YFP using an antibody against YFP and stained with DAPI. The nuclei were stained with DAPI. The stained cross sections were examined under a microscope to assess the presence of staining. Cells which stained positive for YFP expression were observed as brown cells. [0080] A small fraction of the cells in the liver cross section from the mouse with the first and second expression cassettes of Example 1 showed YFP expression. The positive control showed strong staining in all of the cells throughout the cross section. None of the cells in the negative control showed activation of YFP expression.

EXAMPLE 8

[0081] This example demonstrates that the expression cassettes of Example 1 (including the reporter gene Rosa-tdTomato instead of the reporter gene eYFP) detect transcriptional slippage in adult mice treated with short hairpin RNA (shRNA) that targets Tcea.

[0082] Transgenic mice were prepared as described in Example 1 with the exception that the reporter gene eYFP was replaced with the reporter gene Rosa-tdTomato.

[0083] The expression cassettes were tested for the ability to detect transcriptional slippage in adult mice by knocking down factors known to affect transcriptional fidelity. Hydrodynamic gene delivery was used to transfect liver cells with shRNA which targets the Transcriptional elongation factor A family (Tceal, Tcea2, and Tcea3). It is expected that knocking down these factors should increase transcriptional slippage and, therefore, reporter activity. The experimental readout of Cre activity is activation of the fluorescent Cre reporter Rosa-tdTomato. When Cre acts upon this reporter, cells express tdTomato and can be visualized fluorescently. Thus far, efficient liver transfection of shRNA constructs has been demonstrated. Reporter activation in the livers of injected mice has been observed.

[0084] After several experiments, it was determined that 4-6 weeks post-shRNA injection is a suitable time to look for reporter activation in the liver. During the first week following hydrodynamic injection, a number of positive cells were observed in the saline-injected control as well as in the experimental animals. Without being bound to a particular theory or mechanism, it is believed that the hydrodynamic technique was causing the background.

[0085] Reporter activity was high in the thymus of these animals. Without being bound to a particular theory or mechanism, it is believed that this background may be due to lymphocyte invasion following damage to the liver caused by the hydrodynamic injection. The background appears to subside over time.

[0086] Preliminary results showed increased reporter activity at 6 weeks post-injection in mice injected with the Tcea shRNA versus control scrambled shRNA. [0087] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0088] The use of the terms "a" and "an" and "the" and "at least one" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The use of the term "at least one" followed by a list of one or more items (for example, "at least one of A and B") is to be construed to mean one item selected from the listed items (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0089] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. An in vitro method of screening a test agent for the ability to increase or decrease transcription errors, the method comprising:

providing test cells, wherein the test cells comprise:

(a) a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(_n)G, wherein n is an integer from 7 to 20 and n is selected to place the DNA sequence of (i) out of the open reading frame by the absence of one nucleotide, and wherein (ii) is upstream of (i); and

(b) a second expression cassette comprising a DNA sequence encoding a reporter gene and comprising a spacer sequence, wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase;

treating the test cells with a test agent;

transcribing the first expression cassette in the treated test cells;

wherein transcribing (ii) of the first expression cassette comprises making (a) an error or (b) no error in transcription of (ii) of the first expression cassette,

wherein making an error in transcription of (ii) of the first expression cassette produces a Cre recombinase transcript in the open reading frame, and the method further comprises expressing active Cre recombinase from the Cre recombinase transcript, catalyzing removal of the spacer sequence in the second expression cassette by the active Cre recombinase, and expressing the reporter gene;

wherein making no error in transcription of (ii) of the first expression cassette produces a transcript out of the open reading frame for the Cre recombinase, wherein the method further comprises expressing no active Cre recombinase, not catalyzing removal of the spacer sequence in the second expression cassette, and not expressing the reporter gene; providing a control cells, wherein the control cells are identical to the test cells except that the control cells are not treated with the test agent;

measuring expression of the reporter gene in the control cells and in the test cells; and comparing the expression of the reporter gene in the control cells to the expression of the reporter gene in the test cells; wherein an increase in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent increases transcription errors;

wherein a lack of an increase in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent does not increase transcription errors;

wherein a decrease in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent decreases transcription errors; and

wherein a lack of a decrease in expression of the reporter gene in the test cells as compared to the control cells indicates that the test agent does not decrease transcription errors.

2. An in vitro method of detecting a transcription error, the method comprising: transcribing a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(_n)G, wherein n is an integer from 7 to 20 and n is selected to place the DNA sequence of (i) out of the open reading frame by the absence of one nucleotide, and wherein (ii) is upstream of (i);

providing a second expression cassette comprising a DNA sequence encoding a reporter gene and comprising a spacer sequence, wherein the spacer sequence is flanked by loxP sites and the spacer sequence is positioned to prevent expression of the reporter gene in the absence of active Cre recombinase;

wherein transcribing the first expression cassette comprises making (a) an error or (b) no error in transcription of (ii) of the first expression cassette,

wherein making no error in transcription of (ii) of the first expression cassette produces a transcript out of the open reading frame for the Cre recombinase, wherein the method further comprises expressing no active Cre recombinase, not catalyzing removal of the spacer sequence in the second expression cassette, and not expressing the reporter gene; detecting expression or lack of expression of the reporter gene; wherein expression of the reporter gene is indicative of a transcription error and a lack of expression of the reporter gene is indicative of a lack of a transcription error.

3. The method of claim 1 or 2, wherein making an error in transcription comprises misincorporating a nucleotide into a transcript of (ii) which the DNA sequence of (ii) does not encode and adding a nucleotide into a transcript of (ii) which the DNA sequence of (ii) does not encode.

4. The method of claim 1 or 2, wherein making an error in transcription comprises (a) adding a nucleotide into a transcript of (ii) which the DNA sequence of (ii) does not encode or (b) deleting two nucleotides from a transcript of (ii) which are encoded in the DNA sequence of (ii).

5. The method of any one of claims 1 -4, comprising detecting expression or lack of expression of the reporter gene in isolated mouse cells.

6. The method of any one of claims 1-4, comprising detecting expression or lack of expression of the reporter gene in isolated human cells.

7. The method of any one of claims 1-4, comprising detecting expression or lack of expression of the reporter gene in an isolated tissue.

8. The method of any one of claims 1-4, comprising detecting expression or lack of expression of the reporter gene in an isolated organ.

9. A collection of expression cassettes, the collection comprising:

(a) a first expression cassette comprising an open reading frame and comprising (i) a DNA sequence encoding Cre recombinase and (ii) a DNA sequence encoding A(„)G, wherein n is an integer from 7 to 20 and n is selected to place the DNA sequence of (i) out of the open reading frame by the absence of one nucleotide, and wherein (ii) is upstream of (i); and

wherein an error in transcription of (ii) of the first expression cassette produces a Cre recombinase transcript in the open reading frame such that active Cre recombinase is expressed and the expressed active Cre recombinase catalyzes removal of the spacer sequence in the second expression cassette so that the reporter gene is expressed; and

wherein a lack of an error in transcription of (ii) of the first expression cassette produces a transcript out of the open reading frame for the Cre recombinase such that no active Cre recombinase is expressed, no active Cre recombinase catalyzes removal of the spacer sequence in the second expression cassette, and the reporter gene is not expressed.

10. The collection of expression cassettes of claim 9, wherein the error in transcription is a misincorporation of a nucleotide into a transcript of (ii) which the DNA sequence of (ii) does not encode and adding a nucleotide into a transcript of (ii) which the DNA sequence of (ii) does not encode.

1 1. The collection of expression cassettes of claim 9, wherein the error in transcription is (a) addition of a nucleotide into a transcript of (ii) which the DNA sequence of (ii) does not encode or (b) deletion of two nucleotides from a transcript of (ii) which are encoded in the DNA sequence of (ii).

12. One or more recombinant expression vectors comprising the collection of expression cassettes of any one of claims 9-1 1.

13. An isolated host cell comprising the collection of expression cassettes of any one of claims 9-1 1 or the one or more recombinant expression vectors of claim 12.

14. The host cell according to claim 13, wherein the host cell is a mouse cell.

15. The host cell according to claim 13, wherein the host cell is a human cell.

16. An isolated population of cells comprising at least one host cell according to any one of claims 13-15.

17. An isolated tissue comprising the collection of expression cassettes of any one of claims 9-1 1, the one or more recombinant expression vectors of claim 12, the host cell of any one of claims 13-15, or the population of claim 16.

18. An isolated organ comprising the collection of expression cassettes of any one of claims 9-11, the one or more recombinant expression vectors of claim 12, the host cell of any one of claims 13-15, the population of claim 16, or the tissue of claim 17.

19. A non-human animal comprising the collection of expression cassettes of any one of claims 9-11 or the one or more recombinant expression vectors of claim 12.

20. The non-human animal of claim 19, wherein the non-human animal is a mouse.

21. The collection of expression cassettes of any one of claims 1 1 - 13 for use in the screening of a test agent for the ability to increase or decrease transcription errors in a non- human animal.

22. The collection of expression cassettes of any one of claims 1 1-13 for use in the detection of a transcription error in a non-human animal.

23. The collection of expression cassettes for the use of claim 21 or 22, wherein the non-human animal is a mouse.