Control Of Endogenous Promoter Research Articles

Reprogramming Human T Cell Function and Specificity With Non–Viral Genome Targeting

TL Roth, C Puig–Saus, R Yu. Nature. 2018;559(7714):405–409 To develop and optimize nonviral CRISPR-Cas9 genome editing for primary T cells and apply this technique in part to correct a pathogenic mutation in a monogenic autoimmune disease. Using CRISPR-Cas9, primary human T cells from healthy donors and a family with monogenic autoimmune disease were engineered ex vivo along with other important proofs of concept. The CRISPR-Cas9 system was first described as a prokaryotic adaptive immune response, and it has since been widely used as a gene editing tool. CRISPR sequences identify homologous areas in the DNA, and Cas9 creates precise double-stranded breaks. Double-stranded breaks are repaired by either nonhomologous end joining or homology-directed repair (HDR). HDR is less error prone, and by using CRISPR-Cas9 with HDR templates, transgenes can be integrated into the targeted genome with precision. In this study, an electroporation method was optimized for ex vivo primary human T cells to allow for uptake of CRISPR-Cas9 ribonucleoprotein complexes and large (>1 kb) double-stranded DNA (dsDNA) templates for HDR. The technique allowed for optimized cell viability along with efficiency under …

A High-Resolution Map of SBP1 Interactomes in Plasmodium falciparum-infected Erythrocytes.

SummaryThe pathogenesis of malaria parasites depends on host erythrocyte modifications that are facilitated by parasite proteins exported to the host cytoplasm. These exported proteins form a trafficking complex in the host cytoplasm that transports virulence determinants to the erythrocyte surface; this complex is thus essential for malaria virulence. Here, we report a comprehensive interaction network map of this complex. We developed authentic, unbiased, highly sensitive proteomic approaches to determine the proteins that interact with a core component of the complex, SBP1 (skeleton-binding protein 1). SBP1 interactomes revealed numerous exported proteins and potential interactors associated with SBP1 intracellular trafficking. We identified several host-parasite protein interactions and linked the exported protein MAL8P1.4 to Plasmodium falciparum virulence in infected erythrocytes. Our study highlights the complicated interplay between parasite and host proteins in the host cytoplasm and provides an interaction dataset connecting dozens of exported proteins required for P. falciparum virulence.

A gene-specific T2A-GAL4 library for Drosophila.

We generated a library of ~1000 Drosophila stocks in which we inserted a construct in the intron of genes allowing expression of GAL4 under control of endogenous promoters while arresting transcription with a polyadenylation signal 3' of the GAL4. This allows numerous applications. First, ~90% of insertions in essential genes cause a severe loss-of-function phenotype, an effective way to mutagenize genes. Interestingly, 12/14 chromosomes engineered through CRISPR do not carry second-site lethal mutations. Second, 26/36 (70%) of lethal insertions tested are rescued with a single UAS-cDNA construct. Third, loss-of-function phenotypes associated with many GAL4 insertions can be reverted by excision with UAS-flippase. Fourth, GAL4 driven UAS-GFP/RFP reports tissue and cell-type specificity of gene expression with high sensitivity. We report the expression of hundreds of genes not previously reported. Finally, inserted cassettes can be replaced with GFP or any DNA. These stocks comprise a powerful resource for assessing gene function.

Humanized Von Willebrand Factor-Glycoprotein Ibα Interaction in Mouse Models of Hemostasis and Thrombosis

Introduction: The interaction between von Willebrand Factor (VWF) and platelet glycoprotein (GP) Ibα is key for initiating the response to vascular injury that leads to hemostasis or, in pathological conditions, may be a cause of thrombosis. VWF binding to GPIbα occurs through the A1 domain (VWFA1) and its role in platelet adhesion and aggregation becomes progressively more important with increasing shear rates, i.e., in arterioles or pathologically stenosed arteries. Owing to the key role in platelet adhesion/aggregation under arterial flow conditions, VWFA1 has been considered an obvious target for antithrombotic intervention. However, efforts to develop this concept have been complicated by the lack of suitable animal models due to species-specificity in VWFA1-GPIb binding. To obviate the problem, we have generated new mouse strains with humanized VWF-GPIb interaction and characterized the resulting phenotypes in experimental ex vivo and in vivo models of hemostasis and thrombosis.Methods: In the human VWF gene, and in the mouse Vwf ortholog, exon 28 encodes domains A1 and A2, including the VWFA1 GPIb-binding site. We generated a knock-in mouse by targeted insertion of human VWF exon 28 (VWFh28) into the mouse Vwf exon28 locus such that mouse platelet GPIbα (M1) interacted with mouse VWF containing human A1 domain (HA); the strain was designated M1HA (Table). These mice were cross-bred with the previously described transgenic strain (mGPIbαnull;hIbαTg) in which human GPIbα is expressed on the platelet surface in the context of the mouse GPIb-IX-V complex (strain designation: H1MA). The resulting strain was thus designated H1HA, with humanized GPIb-VWF interaction. The unmodified wild type mouse strain used for reference was designated M1MA (Table). All mouse strains were in C57BL/6 genetic background. VWF plasma concentration was measured by ELISA and function ex vivo was evaluated by ristocetin-induced platelet aggregation. In vivo, we measured the tail bleeding time to gauge hemostatic efficiency as well as the propensity to support pathological thrombosis in the carotid artery injured by exposure to ferric chloride.Results: Plasma of VWFh28 mice expressing mouse or human platelet GPIbα had VWF levels (M1HA: 876.4 ± 209.5 mU/ml, n = 16; H1HA: 848.9 ± 121.0 mU/ml, n = 15) not significantly different from wild type mice (M1MA: 1022 ± 267.4 mU/ml, n = 23). Addition of 1.5 mg/ml ristocetin into platelet-rich plasma (PRP) from M1MA, M1HA and H1MA mice elicited no response; only in PRP of H1HA mice did ristocetin cause platelet aggregation that, as in human PRP, was inhibited by the anti-human VWFA1 monoclonal antibody, NMC-4. The tail bleeding time was abnormally prolonged in the M1HA strain expressing human VWFA1/mouse GPIbα, but normal in the H1HA strain expressing human VWFA1/human GPIbα (6.0 ± 3.8 min vs. 1.5 ± 0.9 min; n = 12); the latter was similar to the bleeding time in the M1MA strain (1.0 ± 0.1 min, n = 10). Thrombus formation (time to occlusion) following a carotid artery injury with 9% FeCl3·6H2O was greatly delayed in the M1HA strain (1760.0 ± 538.5 s, n = 6) but similar to WT M1MA in the H1HA strain (485.2 ± 63.7 s vs. 598.3 ± 84.0 s, n = 6, respectively).Conclusions: Knock-in of human VWF exon 28 into the mouse Vwf locus led to successful biosynthesis of chimeric mouse-human VWF protein in vivo under endogenous promoter control, maintaining normal physiologic expression in endothelial cells and megakaryocytes/platelets. Tail bleeding time and in vivo thrombosis assays confirmed the normal functional interaction of mouse VWF containing human A1 domain with hGPIbα. Moreover, the normal response to ristocetin with platelet aggregation, and inhibition of the latter by the specific NMC4 antibody, indicates that the "humanized" H1HA mouse strain could be a powerful tool to select and develop new interventions for the diagnosis and treatment of hemostatic and thrombotic disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9.

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens.

Tumour cells expressing single VEGF isoforms display distinct growth, survival and migration characteristics.

Vascular endothelial growth factor-A (VEGF) is produced by most cancer cells as multiple isoforms, which display distinct biological activities. VEGF plays an undisputed role in tumour growth, vascularisation and metastasis; nevertheless the functions of individual isoforms in these processes remain poorly understood. We investigated the effects of three main murine isoforms (VEGF188, 164 and 120) on tumour cell behaviour, using a panel of fibrosarcoma cells we developed that express them individually under endogenous promoter control. Fibrosarcomas expressing only VEGF188 (fs188) or wild type controls (fswt) were typically mesenchymal, formed ruffles and displayed strong matrix-binding activity. VEGF164- and VEGF120-producing cells (fs164 and fs120 respectively) were less typically mesenchymal, lacked ruffles but formed abundant cell-cell contacts. On 3D collagen, fs188 cells remained mesenchymal while fs164 and fs120 cells adopted rounded/amoeboid and a mix of rounded and elongated morphologies respectively. Consistent with their mesenchymal characteristics, fs188 cells migrated significantly faster than fs164 or fs120 cells on 2D surfaces while contractility inhibitors accelerated fs164 and fs120 cell migration. VEGF164/VEGF120 expression correlated with faster proliferation rates and lower levels of spontaneous apoptosis than VEGF188 expression. Nevertheless, VEGF188 was associated with constitutively active/phosphorylated AKT, ERK1/2 and Stat3 proteins. Differences in proliferation rates and apoptosis could be explained by defective signalling downstream of pAKT to FOXO and GSK3 in fs188 and fswt cells, which also correlated with p27/p21 cyclin-dependent kinase inhibitor over-expression. All cells expressed tyrosine kinase VEGF receptors, but these were not active/activatable suggesting that inherent differences between the cell lines are governed by endogenous VEGF isoform expression through complex interactions that are independent of tyrosine kinase receptor activation. VEGF isoforms are emerging as potential biomarkers for anti-VEGF therapies. Our results reveal novel roles of individual isoforms associated with cancer growth and metastasis and highlight the importance of understanding their diverse actions.

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

BackgroundProtein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels.ResultsUsing a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners.ConclusionOur results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development.

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro(71) and Chro(612) have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.

SR Protein Family Members Display Diverse Activities in the Formation of Nascent and Mature mRNPs In Vivo

The SR proteins are a family of pre-mRNA splicing factors with additional roles in gene regulation. To investigate individual family members in vivo, we generated a comprehensive panel of stable cell lines expressing GFP-tagged SR proteins under endogenous promoter control. Recruitment of SR proteins to nascent FOS RNA was transcription dependent and RNase sensitive, with unique patterns of accumulation along the gene specified by the RNA recognition motifs (RRMs). In addition, all SR protein interactions with Pol II were RNA dependent, indicating that SR proteins are not preassembled with Pol II. SR protein interactions with RNA were confirmed in situ by FRET/FLIM. Interestingly, SC35-GFP also exhibited FRET with DNA and failed to associate with cytoplasmic mRNAs, whereas all other SR proteins underwent nucleocytoplasmic shuttling and associated with specific nuclear and cytoplasmic mRNAs. Because different constellations of SR proteins bound nascent, nuclear, and cytoplasmic mRNAs, mRNP remodeling must occur throughout an mRNA's lifetime.

Inducible Expression of Leukemia-Associated Shp2 (Ptpn11) Affects Multiple Stages of Hematopoiesis and Causes a Fatal Myeloproliferative Disorder (MPD) in Mice

Germ-line mutations in ptpn11, which encodes the protein tyrosine phosphatase Shp2, cause ∼50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ∼35% of sporadic JMML; nearly all other cases have either activating Ras mutations or homozygous Nf1 deficiency. Shp2 mutations are also found at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and B-cell acute lymphoblastic leukemia (B-ALL). NS and leukemia-associated Shp2 mutations can affect the same residue, but result in different substitutions. We showed previously that the leukemia-associated mutants E76K or D61Y, but not wild type (WT), Shp2 transform bone marrow (BM) or fetal liver cells. Transplantation of E76K- or D61Y-transduced BM evoked invasive MPD in ∼60% of recipients, with most dying at 6–7 months. In this model, mutant Shp2 is expressed under retroviral promoter control and the phenotype is variable and incompletely penetrant. To assess the effect of expressing a leukemogenic Shp2 allele under endogenous promoter control, we generated knock-in mice that can conditionally express Shp2D61Y (LSL-Shp2D61Y). Global expression of the D61Y heterozygous allele was embryonic lethal. Post-natal expression of D61Y, induced by treating Mx-1 Cre; LSL-Shp2D61Y mice with pI-pC, evoked fatal MPD with ∼50% mice dying within 5–7 months. Mutant mice showed a marked increase in WBC, expansion of the Gr-1+/Mac-1+population in BM and spleen, and histopathological evidence of infiltrating MPD. Unlike in our retroviral transduction/transplant model (and similar to JMML patients), most induced LSL-Shp2D61Y mice were also anemic. Mutant mice exhibited a marked depletion of quiescent LSK (Lin−Sca1+cKit+) cells in the BM, with a concomitant increase in LSK cells in the spleen. Interestingly, Shp2 mutant-expressing BM failed to promote long term reconstitution in BM transplant assays. Cells from mutant spleens did have some long term multi-lineage reconstitution activity, but the level was substantially less than predicted by their LSK content. Moreover, no transplant recipients develop MPD. In contrast to these effects on the stem cell compartment, D61Y directly allowed cytokine-independent differentiation of CMP and GMP in vitro. Macrophages from mutant mice showed enhanced GM-CSF-mediated proliferation and ERK activation. Our results showed that expression of leukemia-associated Shp2 at endogenous levels is sufficient to evoke MPD and has cell type-specific effect on different stages of hematopoiesis.

Activating Shp2 Mutations Cause Progressive Myeloproliferative Disorder in Mice.

Mutations in the SH2-containing tyrosine phosphatase, Shp2 (PTPN11) have been found in human diseases. Germ-line Shp2 mutations cause ~50% of Noonan Syndrome (NS), which is associated with an increased risk of juvenile myelomonocytic leukemia (JMML). Somatic Shp2 mutations are found in ~35% of sporadic JMML, most other cases have either activating Ras mutations or homozygous Nf1 deficiency. Shp2 mutations are also found at lower incidence in acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and B-cell acute lymphoblastic leukemia (B-ALL). NS and leukemia-associated Shp2 mutations often affect the same residue, but result in different substitution. We previously showed that leukemia-associated mutants E76K or D61Y, but not wild type (WT) Shp2 could transform bone marrow (BM) or fetal liver cells and give rise to cytokine-independent myeloid colony formation. Transplantation of E76K- or D61Y- (but not parental virus or WT Shp2) transduced BM evoked invasive MPD in most recipients and majority of them died at 6–7 months. We have generated knock-in mice for the Shp2 mutants D61G and D61Y to better understand the effects of these mutations under endogenous promoter control. Whereas D61G is associated with both NS and leukemia, D61Y is only found in leukemia and catalytically more active than D61G in basal state. D61G/+ mice exhibited all major features of NS and a mild, well-tolerated MPD for ~1 year and then progressed to fatal MPD. Constitutive expression of the D61Y heterozygous allele was embryonic lethal. However, when an inducible allele of D61Y (inD61Y) was crossed to Mx-Cre mice and expressed upon pI-pC induction, these mice rapidly developed MPD and ~50% mice died within a period of 4–7 months. Immunophenotypic analysis of bone marrow and spleen of these mice showed marked expansion of Gr-1/Mac-1 positive population indicating that these mice died of infiltrating MPD. Bone marrow macrophages from D61G/+ and inD61Y mice showed increased GM-CSF-evoked proliferation and increased overall tyrosine phosphorylation. Likewise GM-CSF-evoked activation of Erk, Akt, Jak2 and Stat5 were enhanced, as was Ras loading suggesting that leukemogenic Shp2 causes MPD by enhancing both Ras/Erk and Jak2/Stat5 pathways. The results of our retroviral and knock-in models suggest that leukemia-associated Shp2 mutant is sufficient to initiate myeloid leukemogenesis in mice.

O4-01-06: Astrocytes have a reduced capacity to secrete apolipoprotein (APO) E4 leading to substantially reduced brain levels

Inheritance of the apolipoprotein E4 allele increases the risk of developing Alzheimer’s disease, however, the molecular mechanisms underlying this association remain elusive. In order to study apoE4 in a physiologically relevant environment we sought to identify human astrocyoma cell lines that endogenously express apoE4. We apoE genotyped a range of human cell-lines and identified two astrocytomas that expressed the apoE4 isoform. In order to determine the relative amounts of apoE4 to total apoE secreted from these cell-lines we developed two specific immunoassays; an assay to measure total apoE irrespective of isoform and an apoE4 selective assay. Surprisingly, and in contrast to the expected equal contribution of each apoE isoform to the total apoE pool, we found that less than 20 % of the total apoE secreted into the media of apoE3/4 astrocytomas was attributed to apoE4. Next, we sought to determine whether the reduced level of apoE4 secretion from astrocytes translated into reduced absolute levels of apoE4 in the brain. We examined apoE protein levels in the brains of 12 week old humanized 3/3, 4/4 and 3/4 knock-in mice, where the apoE expression is under the control of endogenous promoters. Importantly, these apoE knock-in mice showed a genotype dependent decrease in cortical apoE levels; 3/3 3/4 4/4. Next, we sought to examine the relative contributions of apoE4 relative to apoE3 in the 3/4 mouse brains. Consistent with the in-vitro astrocyte data, apoE4 represented less than 20 % of the total cortical apoE. Moreover, the absolute amount of apoE3 per allele is similar between 3/3 and 3/4 mice, implying that the reduced levels of total apoE in 3/4 knock-in mice can be fully explained by the reduction in apoE4 levels. Taken together these data suggest that under physiological relevant conditions, astrocytes have a reduced capacity to secrete apoE4 that ultimately leads to substantially reduced brain apoE levels. Moreover, the genotype dependent decrease in cortical apoE levels observed in this study, mirror the relative risk of developing Alzheimer’s disease and suggest that low levels of total apoE exhibited by 4 carriers may directly contribute to the disease progression.

Proteomic Analysis of SRm160-containing Complexes Reveals a Conserved Association with Cohesin

In this study, we describe a rapid immunoaffinity purification procedure for gel-free tandem mass spectrometry-based analysis of endogenous protein complexes and apply it to the characterization of complexes containing the SRm160 (serine/arginine repeat-related nuclear matrix protein of 160 kDa) splicing coactivator. In addition to promoting splicing, SRm160 stimulates 3'-end processing via its N-terminal PWI nucleic acid-binding domain and is found in a post-splicing exon junction complex that has been implicated in coupling splicing with mRNA turnover, export, and translation. Consistent with these known functional associations, we found that the majority of proteins identified in SRm160-containing complexes are associated with pre-mRNA processing. Interestingly, SRm160 is also associated with factors involved in chromatin regulation and sister chromatid cohesion, specifically the cohesin subunits SMC1alpha, SMC3, RAD21, and SA2. Gradient fractionation suggested that there are two predominant SRm160-containing complexes, one enriched in splicing components and the other enriched in cohesin subunits. Co-immunoprecipitation and co-localization experiments, as well as combinatorial RNA interference in Caenorhabditis elegans, support the existence of conserved and functional interactions between SRm160 and cohesin.

Development of a Keratinocyte-Based Screening Model for Antipsoriatic Drugs Using Green Fluorescent Protein Under the Control of an Endogenous Promoter

Inflamed epidermis (psoriasis, wound healing, ultraviolet-irradiated skin) harbors keratinocytes that are hyperproliferative and display an abnormal differentiation program. A distinct feature of this so-called regenerative maturation pathway is the expression of proteins such as the cytokeratins CK6, CK16, and CK17 and the antiinflammatory protein SKALP/elafin. These proteins are absent in normal skin but highly induced in lesional psoriatic skin. Expression of these genes can be used as a surrogate marker for psoriasis in drug-screening procedures of large compound libraries. The aim of this study was to develop a keratinocyte cell line that contained a reporter gene under the control of a psoriasis-associated endogenous promoter and demonstrate its use in an assay suitable for screening. We generated a stably transfected keratinocyte cell line that expresses enhanced green fluorescent protein (EGFP), under the control of a 0.8-kb fragment derived from the promoter of the SKALP/elafin gene, which confers high levels of tissue-specific expression at the mRNA level. Induction of the SKALP promoter by tumor necrosis factor-alpha resulted in increased expression levels of the secreted SKALP-EGFP fusion protein as assessed by direct readout of fluorescence and fluorescence polarization in 96-well cell culture plates. The fold stimulation of the reporter gene was comparable to that of the endogenous SKALP gene as assessed by enzyme-linked immunosorbent assay. Although the dynamic range of the screening system is limited, the small standard deviation yields a Z factor of 0.49. This indicates that the assay is suitable as a high-throughput screen, and provides proof of the concept that a secreted EGFP fusion protein under the control of a physiologically relevant endogenous promoter can be used as a fluorescence-based high-throughput screen for differentiation-modifying or antiinflammatory compounds that act via the keratinocyte.

Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis

The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis.

The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

Modifying the mammalian genome by gene targeting.

Over the past year, gene targeting in mammalian cells has become a facile technology. By using a variety of selection and screening protocols, it has become possible to direct modifications at the nucleotide level to specific genes, to target marker genes so that they become expressed under the control of endogenous promoters and to delete large regions of the genome.