Cotranslational Cleavage Research Articles

Multimodal mechanisms of pathogenic variants in the signal peptide of FIX leading to hemophilia B

AbstractSignal peptide (SP) is essential for protein secretion, and pathogenic variants in the SP of factor IX (FIX) have been identified in hemophilia B (HB). However, the underlying mechanism for the genotype-phenotype correlation of these variants has not been well studied. Here, we systematically examined the effects of 13 pathogenic point variants in the SP of FIX using different approaches. Our results showed that these point variants lead to HB by missense variants and/or aberrant premessenger RNA (pre-mRNA) splicing. The missense variants in a hydrophobic core (h-region) mainly affected the cotranslational translocation function of the SP, and those in C-terminal containing cleavage site (c-region) caused FIX deficiency mainly by disturbing the cotranslational translocation and/or cleavage of the SP. Almost absolute aberrant pre-mRNA splicing was only observed in variants of c.82T>G, but a slight change of splicing patterns was found in variants of c.53G>T, c.77C>A, c.82T>C, and c.83G>A, indicating that these variants might have different degrees of impact on pre-mRNA splicing. Although two 6-nt deletion aberrant pre-mRNA splicing products caused FIX deficiency by disturbing the SP cleavage, they could produce some functional mature FIX, and vitamin K could increase the secretion of functional FIX. Taken together, our data indicated that pathogenic variants in the SP of FIX caused HB through diverse molecular mechanisms or even a mixture of several mechanisms, and vitamin K availability could be partially attributed to varying bleeding tendencies in patients carrying the same variant in the SP.

Generation of an INSULIN-H2B-Cherry reporter human iPSC line.

Differentiating human induced pluripotent stem cells (hiPSCs) into insulin (INS)-producing β-like cells has potential for diabetes research and therapy. Here, we generated a heterozygous fluorescent hiPSC reporter, labeling INS-producing β-like cells. We used CRISPR/Cas9 technology to knock-in a T2A-H2B-Cherry cassette to replace the translational INS stop codon, enabling co-transcription and T2A-peptide mediated co-translational cleavage of INS-T2A and H2B-Cherry. The hiPSC-INS-T2A-H2B-Cherry reporter cells were pluripotent and showed multi-lineage differentiation potential. Cells expressing the β-cell specific hormone INS are identified by nuclear localized H2B-Cherry reporter upon pancreatic endocrine differentiation. Thus, the generated reporter hiPSCs enable live identification of INS hormone-producing β-like cells.

Downregulation of the Arg/N-degron Pathway Sensitizes Cancer Cells to Chemotherapy In Vivo

The N-degron pathway is an emerging target for anti-tumor therapies, because of its capacity to positively regulate many hallmarks of cancer, including angiogenesis, cell proliferation, motility, and survival. Thus, inhibition of the N-degron pathway offers the potential to be a highly effective anti-cancer treatment. With the use of a small interfering RNA (siRNA)-mediated approach for selective downregulation of the four Arg/N-degron-dependent ubiquitin ligases, UBR1, UBR2, UBR4, and UBR5, we demonstrated decreased cell migration and proliferation and increased spontaneous apoptosis in cancer cells. Chronic treatment with lipid nanoparticles (LNPs) loaded with siRNA in mice efficiently downregulates the expression of UBR-ubiquitin ligases in the liver without any significant toxic effects but engages the immune system and causes inflammation. However, when used in a lower dose, in combination with a chemotherapeutic drug, downregulation of the Arg/N-degron pathway E3 ligases successfully reduced tumor load by decreasing proliferation and increasing apoptosis in amouse model of hepatocellular carcinoma, while avoiding theinflammatory response. Our study demonstrates that UBR-ubiquitin ligases of the Arg/N-degron pathway are promising targets for the development of improved therapies for many cancer types.

Continuous translation of circularized mRNA improves recombinant protein titer

Recent success in demonstrating the translation of circular RNA open reading frames or circular mRNA, may offer a new avenue for improving recombinant protein production from cell and cell-free expression platforms. Initiation and termination are two rate limiting steps of translation. Circular RNA as a class of RNA is defined by covalent joining of terminal ends to give a closed loop structure. By encoding a gene lacking a stop codon on a circular RNA molecule an infinite open reading frame is generated permitting continuously translating circular mRNA (CTC mRNA). CTC mRNAs have shown promise in enhancing the production of multimeric polyproteins in bacterial cell-free expression systems. Problems arise when homogenous, functional post-translationally modified protein is required. To produce post-translationally modified, secreted protein from an CTC mRNA we investigated co-translational cleavage of nascent polypeptide chains by incorporating a 2 A “self-cleavage” peptide motif. Using a model recombinant human glycoprotein Erythropoietin (EPO) we demonstrate for the first time the ability to produce secreted protein from an infinite circular mRNA in live mammalian cells. Both cell-specific and volumetric productivity were improved by using circular mRNAs. This study pioneers the potential of recombinant protein production from CTC mRNA in mammalian cells.

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

Abstract 1614: Intratumoral delivery of a P2A-linked bicistronic IL-12 construct leads to high intratumoral expression and systemic anti-tumor response

Abstract The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines can result in serious adverse effects, often life-threatening. DNA transfer via electroporation (EP) is a safe and effective method of delivering cytokines to target tissues. Intratumoral (IT) electroporation of Interleukin 12 (IL-12), a potent immunomodulatory cytokine, is well tolerated with an acceptable safety profile and objective response rates of 34-40% and 33% in Phase II clinical trials in advanced melanoma and Merkel cell carcinoma, respectively. We sought to improve the systemic anti-tumor response of IT pIL-12-EP by improving IL-12p70 expression and electroporation conditions, which were evaluated in vitro and in vivo with a two-tumor syngeneic mouse model of melanoma. As functional IL-12 p70 is a heterodimer, we compared different expression constructs to achieve high levels of IL-12 protein expression. IL-12p70 protein expression from a plasmid that incorporated a picornavirus-derived co-translational cleavage site (P2A) was higher than constructs with an internal ribosomal entry sequence (IRES) or a fusion of the p35 and p40 subunits. In functional in vitro assays, pIL-12(P2A) was superior to pIL-12(IRES) and the fusion protein. Using the murine B16.F10 tumor model, we show that IT EP of pIL-12 (P2A) plasmid regresses the treated lesions in a dose-dependent manner compared to control treatments. Systemic effects of IT-expressed IL-12 was assessed by monitoring generation of an antigen-specific CD8 T cell response and regression of B16.F10 contralateral (untreated) tumors following primary tumor electroporation. IT-pIL12-EP treatment with the P2A-linked construct resulted in a significant increase in antigen-specific CD8 T cells, as well as enhanced contralateral tumor growth inhibition suggesting the induction of strong systemic anti-tumor immune response. * Contributed equally to this work Citation Format: David A. Canton, Christoph Burkart, Shawna A. Shirley, Anandaroop Mukhopadhyay, Richard J. Connolly, Arya Bahrami, Jean S. Campbell, Robert H. Pierce. Intratumoral delivery of a P2A-linked bicistronic IL-12 construct leads to high intratumoral expression and systemic anti-tumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1614. doi:10.1158/1538-7445.AM2017-1614

Widespread pre-translational regulation of the inclusion of signal peptides in human proteins

Signal peptides (SP) are cleavable N-terminal protein motifs used co-translationally for entry of nascent polypeptides into the secretory pathway. Their co-translational cleavage prevents their extensive post-translational regulation and flexibility in their usage is made possible by the control of their inclusion at a pre-translational level. To characterize this regulation on a transcriptome scale, we analyzed the level and mechanisms of inclusion of the 3298 most likely human SP-encoding genes, 47% of which alternatively express their SP. Analysis of RNA-seq data across different normal human tissues indicates that pre-translational regulation of the SP differs depending on tissue-coverage of the gene, with alternative SP genes more likely to be widely expressed than constitutive SP genes. SP inclusion represents a new metric to measure functional gene expression and its deregulation in disease. Our analysis supports the extensive use of pre-translational regulation of SP inclusion, with functional consequences and implications for biomarker discovery.

Balanced secretion of anti-CEA \xd7 anti-CD3 diabody chains using the 2A self-cleaving peptide maximizes diabody assembly and tumor-specific cytotoxicity

Adoptive transfer of genetically engineered human cells secreting bispecific T-cell engagers has shown encouraging therapeutic effects in preclinical models of cancer. However, reducing the toxicity and improving the effectiveness of this emerging immunotherapeutic strategy will be critical to its successful application. We have demonstrated that for gene-based bispecific antibody strategies, two-chain diabodies have a better safety profile than single-chain tandem scFvs (single-chain variable fragments), because their reduced tendency to form aggregates reduces the risk of inducing antigen-independent T-cell activation. Here, we demonstrate that the incorporation of a 2A self-processing peptide derived from foot-and-mouth disease virus conveying co-translational cleavage into a two-chain anti-CD3 × anti-CEA diabody gene enables near-equimolar expression of diabody chains 1 and 2, and thus increases the final amount of assembled diabody. This was found to maximize diabody-mediated T-cell activation and cytotoxicity against carcinoembryonic antigen-positive tumor cells.

Comparing lamin proteins post-translational relative stability using a 2A peptide-based system reveals elevated resistance of progerin to cellular degradation

ABSTRACTNuclear lamins are the major components of the nuclear lamina at the periphery of the nucleus, supporting the nuclear envelope and participating in many nuclear processes, including DNA replication, transcription and chromatin organization. A group of diseases, the laminopathies, is associated with mutations in lamin genes. One of the most striking cases is Hutchinson-Gilford progeria syndrome (HGPS) which is the consequence of a lamin A dominant negative mutant named progerin. Due to the abnormal presence of a permanent C-terminal farnesyl tail, progerin gradually accumulates on the nuclear membrane, perturbing a diversity of signalings and transcriptional events. The accumulation of progerin has led to the speculation that progerin possesses higher stability than the wild type lamin A protein. However, the low solubility of lamin proteins renders traditional immunoprecipitation-dependent methods such as pulse-chase analysis ineffective for comparing the relative stabilities of mutant and wild type lamins. Here, we employ a novel platform for inferring differences in lamin stability, which is based on normalization to a co-translated reporter protein following porcine teschovirus-1 2A peptide-mediated co-translational cleavage. The results obtained using this method support the notion that progerin is more stable than lamin A. Moreover, treatment of FTI reduces progerin relative stability to the level of wild type lamin A.

Spiroepoxytriazoles Are Fumagillin-like Irreversible Inhibitors of MetAP2 with Potent Cellular Activity.

Methionine aminopeptidases (MetAPs) are responsible for the cotranslational cleavage of initiator methionines from nascent proteins. The MetAP2 subtype is up-regulated in many cancers, and selective inhibition of MetAP2 suppresses both vascularization and growth of tumors in animal models. The natural product fumagillin is a selective and potent irreversible inhibitor of MetAP2, and semisynthetic derivatives of fumagillin have shown promise in clinical studies for the treatment of cancer, and, more recently, for obesity. Further development of fumagillin derivatives has been complicated, however, by their generally poor pharmacokinetics. In an attempt to overcome these limitations, we developed an easily diversifiable synthesis of a novel class of MetAP2 inhibitors that were designed to mimic fumagillin's molecular scaffold but have improved pharmacological profiles. These substances were found to be potent and selective inhibitors of MetAP2, as demonstrated in biochemical enzymatic assays against three MetAP isoforms. Inhibitors with the same relative and absolute stereoconfiguration as fumagillin displayed significantly higher activity than their diastereomeric and enantiomeric isomers. X-ray crystallographic analysis revealed that the inhibitors covalently modify His231 in the MetAP2 active site via ring-opening of a spiroepoxide. Biochemically active substances inhibited the growth of endothelial cells and a MetAP2-sensitive cancer cell line, while closely related inactive isomers had little effect on the proliferation of either cell type. These effects correlated with altered N-terminal processing of the protein 14-3-3-γ. Finally, selected substances were found to have improved stabilities in mouse plasma and microsomes relative to the clinically investigated fumagillin derivative beloranib.

Synthetic Biology Tools for Bioprospecting of Natural Products in Eukaryotes

Filamentous fungi have the capacity to produce a battery of natural products of often unknown function, synthesized by complex metabolic pathways. Unfortunately, most of these pathways appear silent, many in intractable organisms, and their products consequently unidentified. One basic challenge is the difficulty of expressing a biosynthesis pathway for a complex natural product in a heterologous eukaryotic host. Here, we provide a proof-of concept solution to this challenge and describe how the entire penicillin biosynthesis pathway can be expressed in a heterologous host. The method takes advantage of a combination of improved yeast invivo cloning technology, generation of polycistronic mRNA for the gene cluster under study, and an amenable and easily manipulated fungal host, i.e., Aspergillus nidulans. We achieve expression from a single promoter of the pathway genes to yield a large polycistronic mRNA by using viral 2A peptide sequences to direct successful cotranslational cleavage of pathway enzymes.

A Protocol to Measure the Extent of Cell-to-cell Movement of RNA Viruses in Planta

Here, we present a simple and rapid protocol to measure the extent of cell-to-cell movement of RNA viruses in planta. To do that, the green fluorescent protein (GFP) gene was incorporated into the genome of Melon necrotic spot virus (MNSV) as a coat protein (CP) fusion protein using the Thosea asigna virus 2A catalytic peptide (TaV 2a) (Serra-Soriano et al., 2014). TaV 2a allows the co-translational cleavage of the fusion protein resulting in the independent expression of both proteins (Kim et al., 2011). Viral infection was initiated by agro-infiltration of Cucumis melo leaves. At 6-7 days post-infiltration, fluorescent infection foci images were taken with a fluorescent stereo microscope and infection areas were measured using FIJI software., 在这里，我们提出一个简单和快速的协议，以测量RNA病毒在植物中的细胞到细胞运动的程度。 为此，将绿色荧光蛋白（GFP）基因作为外壳蛋白（CP）融合蛋白掺入到Melon坏死斑病毒（MNSV）的基因组中，使用Sucha asigna病毒/Ta> 2A催化肽（TaV 2a）（Serra-Soriano等人，2014）。 TaV 2a允许融合蛋白的共翻译切割，导致两种蛋白的独立表达（Kim等人，2011）。 病毒感染通过对黄瓜叶的农杆菌浸润来启动。 在浸润后6-7天，用荧光立体显微镜拍摄荧光感染灶图像，并使用FIJI软件测量感染面积。

The Sox17CreERT2 knock‐in mouse line displays spatiotemporal activation of Cre recombinase in distinct Sox17 lineage progenitors

The HMG-box transcription factor Sox17 is essential for endoderm formation, vascular development, and definitive hematopoiesis. To investigate the fate of distinct Sox17-expressing progenitor cells in a spatiotemporal manner, we generated a hormone-inducible CreERT2 knock-in mouse line. By homologous recombination we fused a codon improved, ligand-dependent estrogen receptor Cre recombinase by an intervening viral T2A sequence for co-translational cleavage to the 3' coding region of Sox17. Induction of Cre activity by administration of tamoxifen at defined time points of early mouse development and subsequent genetic lineage tracing confirmed the inducibility and tissue specificity of Cre recombination. Furthermore, Cre activity could be selectively induced in extra-embryonic and embryonic endoderm lineages, the primitive gut tube, and in endothelial cells of the vascular system as well as in the hemogenic endothelium of the dorsal aorta. The Sox17CreERT2 mouse line therefore represents a new tool for genetic lineage tracing in a tissue-specific manner and in addition enables lineage-restricted functional analysis.

The protein coexpression problem in biotechnology and biomedicine: virus 2A and 2A-like sequences provide a solution

Synthetic biology enables us to create genes virtually at will. Ensuring that multiple genes are efficiently coexpressed within the same cell in order to assemble multimeric complexes, transfer biochemical pathways and transfer traits is more problematic. Viruses such as picornaviruses accomplish exactly this task: they generate multiple different proteins from a single open reading frame. The study of how foot-and-mouth disease virus controls its protein biogenesis led to the discovery of a short oligopeptide sequence, ‘2A’, that is able to mediate a cotranslational cleavage between proteins. 2A and ‘2A-like’ sequences (from other viruses and cellular sequences) can be used to concatenate multiple gene sequences into a single gene, ensuring their coexpression within the same cell. These sequences are now being used in the treatment of cancer, in the production of pluripotent stem cells, and to create transgenic plants and animals among a host of other biotechnological and biomedical applications.

KHARON1 Mediates Flagellar Targeting of a Glucose Transporter in Leishmania mexicana and Is Critical for Viability of Infectious Intracellular Amastigotes

The LmxGT1 glucose transporter is selectively targeted to the flagellum of the kinetoplastid parasite Leishmania mexicana, but the mechanism for targeting this and other flagella-specific membrane proteins among the Kinetoplastida is unknown. To address the mechanism of flagellar targeting, we employed in vivo cross-linking, tandem affinity purification, and mass spectrometry to identify a novel protein, KHARON1 (KH1), which is important for the flagellar trafficking of LmxGT1. Kh1 null mutant parasites are strongly impaired in flagellar targeting of LmxGT1, and trafficking of the permease was arrested in the flagellar pocket. Immunolocalization revealed that KH1 is located at the base of the flagellum, within the flagellar pocket, where it associates with the proximal segment of the flagellar axoneme. We propose that KH1 mediates transit of LmxGT1 from the flagellar pocket into the flagellar membrane via interaction with the proximal portion of the flagellar axoneme. KH1 represents the first component involved in flagellar trafficking of integral membrane proteins among parasitic protozoa. Of considerable interest, Kh1 null mutants are strongly compromised for growth as amastigotes within host macrophages. Thus, KH1 is also important for the disease causing stage of the parasite life cycle.

Ubiquitin Reference Technique and Its Use in Ubiquitin-Lacking Prokaryotes

In a pulse-chase assay, the in vivo degradation of a protein is measured through a brief labeling of cells with, for example, a radioactive amino acid, followed by cessation of labeling and analysis of cell extracts prepared at different times afterward (“chase”), using immunoprecipitation, electrophoresis and autoradiography of a labeled protein of interest. A conventional pulse-chase assay is fraught with sources of data scatter, as the efficacy of labeling and immunoprecipitation can vary, and sample volumes can vary as well. The ubiquitin reference technique (URT), introduced in 1996, addresses these problems. In eukaryotes, a DNA-encoded linear fusion of ubiquitin to another protein is cleaved by deubiquitylases at the ubiquitin-protein junction. A URT assay uses a fusion in which the ubiquitin moiety is located between a downstream polypeptide (test protein) and an upstream polypeptide (a long-lived reference protein). The cotranslational cleavage of a URT fusion by deubiquitylases after the last residue of ubiquitin produces, at the initially equimolar ratio, a test protein with a desired N-terminal residue and a reference protein containing C-terminal ubiquitin moiety. In addition to being more accurate than pulse-chases without a reference, URT makes it possible to detect and measure the degradation of a test protein during the pulse (before the chase). Because prokaryotes, including Gram-negative bacteria such as, for example, Escherichia coli and Vibrio vulnificus, lack the ubiquitin system, the use of URT in such cells requires ectopic expression of a deubiquitylase. We describe designs and applications of plasmid vectors that coexpress, in bacteria, both a URT-type fusion and Ubp1, a deubiquitylase of the yeast Saccharomyces cerevisiae. This single-plasmid approach extends the accuracy-enhancing URT assay to studies of protein degradation in prokaryotes.

Stochastic Properties of Transcription Factor Expression Revealed by Single-Molecule Noise Analysis

Transcription factors (TFs) play important roles in gene regulation. Monitoring TF production in real time will help to elucidate how the stochasticity in the expression TFs influences the expression of their target genes. However, many TFs are expressed at low levels and are thus difficult to detect. Further, labeling TFs with fluorescent proteins can potentially disrupt their regulatory functions. Here, we developed a novel strategy, Co-Translational Activation by Cleavage (CoTrAC), to monitor the stochastic expression of a TF, λ repressor CI, in live E. coli cells at the single-molecule level. CI becomes fully functional upon co-translational cleavage from a membrane-targeted reporter, which can be counted individually. Using this strategy, we monitored the production of CI in real time in the regulatory context of the λ phage genetic switch. We developed a robust noise analysis to decompose the total noise in CI production into intrinsic, extrinsic and correlation sources. We show that intrinsic noise likely arises from both transcriptional and translational bursting. Extrinsic noise accounts for most of the total noise on the time scale of one cell cycle. CI enhances transcription primarily by increasing bursting frequency. Furthermore, negative autoregulation increases intrinsic noise, but counteracts extrinsic noise and ensures that molecular memory diminishes on a time scale shorter than one cell cycle. The experimental and theoretical approaches presented here offer a unique opportunity to investigate noise in gene regulatory networks.

FltpT2AiCre: A new knock-in mouse line for conditional gene targeting in distinct mono- and multiciliated tissues

We recently identified Flattop (Fltp; 1700009p17Rik) in a screen for potential Foxa2 target and novel mouse organizer genes. Besides its expression in the embryonic node, we found that Fltp is active in other monociliated tissues such as the sensory organs of the inner ear, duct and islets of the pancreas as well as in testis. Additionally, Fltp mRNA is expressed in multiciliated epithelial cells of the lung and of the choroid plexi in the brain. To genetically lineage trace these cells during development and injury as well as to conditionally inactivate genes in these tissues, we generated a Cre recombinase knock-in mouse line using the Fltp gene locus. By homologous recombination we have fused the Fltp open-reading frame to a tandem affinity purification (TAP) tag followed by an intervening viral T2A sequence for co-translational cleavage and an improved Cre recombinase (iCre). This strategy allows both the analysis of the tagged Fltp-TAP-T2A protein and the usage of the iCre recombinase for conditional targeting approaches. Using the ROSA26 reporter mouse line we show that FltpT2AiCre is first active in the monociliated cells of the node, notochord, floorplate and prechordal plate, consistent with the Fltp-TAP-T2A protein production in the node progenitor cells. Furthermore iCre recombinase activity is detected in multiciliated tissues such as choroid plexi of the brain and epithelial cells of the lung with the onset at E10.5 and E13.5, respectively. In the pancreas, β-galactosidase activity is seen in the monociliated cells of the pancreatic duct and islet of Langerhans. Intercrossing FltpT2AiCre mice with the CAG-CAT-EGFP reporter mouse line further confirms iCre activity in multiciliated cells of the lung and brain on a cellular level. Thus, the FltpT2AiCre line is a powerful tool to conditionally inactivate genes in distinct mono- and multiciliated tissues and to analyze the tagged Fltp protein in vivo.

Transcription Activation via Transcriptional Bursting

Transcription factors (TFs) often regulate their own expression; monitoring TF production in real time will help elucidate autoregulatory mechanisms. However, many TFs are expressed at low levels and are difficult to detect. Further, labeling TFs with fluorescent proteins risks disrupting their regulatory functions. Here, we report a novel strategy, Co-Translational Activation by Cleavage (CoTrAC), to monitor autoregulation of a transcription factor, λ repressor cI, in live E. coli cells. CI becomes fully functional upon co-translational cleavage from a membrane-targeted fluorescent reporter, which can be counted individually. Using this strategy, we discovered that cI activates its own expression via transcriptional bursting_the production of multiple mRNA molecules in brief, well-separated periods of time. CI increases not only the frequency but also the size of transcriptional bursts. Negative autoregulation by cI decreases the burst frequency, but not the burst size. These results link activation of gene expression by a TF to burst-like transcription.

S1684 Sprouty-2 Enhances Cell Proliferation and Induces Resistance to Apoptosis Through IGF-1R Pathway in Colon Cancer Cells

Background: The Caudal-related homeobox (Cdx) transcription factor Cdx2 is a homeodomain protein related to the Drosophila Caudal gene. It is an important regulator of intestinespecific gene expression and columnar morphogenesis. It plays a key role in intestinal cell differentiation, cell to cell adhesion, columnar morphogenesis, and proliferation. Cdx2 has been implicated in a number of human disease processes including colon cancer and intestinal metaplasia. The purpose of this study is to develop a Cre-regulated Cdx2 expression model to facilitate the study of these complex and multi-staged processes. Other inducible systems generally yield a leaky expression that can blunt the Cdx2 response. A Cre-inducible system would not share this limitation. Methods: We have subcloned a Flag-Cdx2 cDNA into a Cre-regulated expression vector. In this construct, Cdx2 is linked in frame to a histone 2BRFP(Cherry) reporter. In between is a picornavirus 2A peptide directing cotranslational cleavage of the linked proteins, thereby releasing the Flag-Cdx2. Upstream to this sequence is a _eta_actin promoter sequence (with CMV enhancer sequence) to drive expression of the bicistronic mRNA. In between these is a loxP flanked cassette that includes a PGK-neo selectable marker and a SV40 virus tpA transcriptional stop sequence. Once integrated, this locus will remain silent until expression of Cre recombinase. This expression vector is cotransfected along with an expression vector for CreER(T2). Subsequent use of Tamoxifen enables drug-dependent control of Cre-mediated DNA recombination. Results: The Creresponsive Cdx2 clone was created and confirmed by DNA sequencing. 293T cells were transiently transfected with this construct, with and without a Cre expression vector. In the absence of Cre recombinase, there was immunofluorescent H2B detected. Conversely, in the presence of Cre co-transfection, reporter immunofluorescent product was strongly observed within cells. In addition, a whole cell lysate was prepared from these cells and analyzed by Western blot using anti-Cdx2 and anti-Flag-Cdx2 probes. Results showed that Flag-Cdx2 was identified exclusively in the Cdx2 clone + Cre co-transfection sample only. Stably transfected HCT116, DLD1, and Colo 205 cells are currently being raised for studies of the effects of Cdx2 induction on cell proliferation and cell to cell adhesion. Conclusions: We have developed a novel Cre-recombinase regulated Cdx2 expression system allowing for the spatiotemporal study of Cdx2 in both In Vitro and In Vivo models. Further studies with stable transfection of our Cdx2-inducible clone into colon cancer cell lines are underway.