Cre Expression Research Articles

NMDA Receptor in Vasopressin 1b Neurons Is Not Required for Short-Term Social Memory, Object Memory or Aggression.

The arginine vasopressin 1b receptor (Avpr1b) plays an important role in social behaviors including aggression, social learning and memory. Genetic removal of Avpr1b from mouse models results in deficits in aggression and short-term social recognition in adults. Avpr1b gene expression is highly enriched in the pyramidal neurons of the hippocampal cornu ammonis 2 (CA2) region. Activity of the hippocampal CA2 has been shown to be required for normal short-term social recognition and aggressive behaviors. Vasopressin acts to enhance synaptic responses of CA2 neurons through a NMDA-receptor dependent mechanism. Genetic removal of the obligatory subunit of the NMDA receptor (Grin1) within distinct hippocampal regions impairs non-social learning and memory. However, the question of a direct role for NMDA receptor activity in Avpr1b neurons to modulate social behavior remains unclear. To answer this question, we first created a novel transgenic mouse line with Cre recombinase knocked into the Avpr1b coding region to genetically target Avpr1b neurons. We confirmed this line has dense Cre expression throughout the dorsal and ventral CA2 regions of the hippocampus, along with scattered expression within the caudate-putamen and olfactory bulb (OB). Conditional removal of the NMDA receptor was achieved by crossing our line to an available floxed Grin1 line. The resulting mice were measured on a battery of social and memory behavioral tests. Surprisingly, we did not observe any differences between Avpr1b-Grin1 knockout mice and their wildtype siblings. We conclude that mice without typical NMDA receptor function in Avpr1b neurons can develop normal aggression as well as short-term social and object memory performance.

Neural crest derived boundary cap cells: A source of pericytes to the developing peripheral vasculature

In addition to Schwann cells (SC) and sensory neurons, two major components of the peripheral nervous system, the neural crest gives rise to boundary cap cells (BC), transiently located at entry and exit points of all embryonic nerves. By performing BCs fate mapping with mice driving Cre expression under control of BC markers, Krox20/Egr2 and Prss56 we have shown that BC derivatives (BCd) migrate into the nerve roots and nerve terminals of the skin. Whereas in the nerve roots, BC derivatives traced with both markers give rise to SC and sensory neurons, in the skin their fate appeared different. Those deriving from Prss56-expressing BCs correspond to SC, the subpopulation of nerve derived melanocytes and neurogenic stem-like cells. In stark contrast, once arrived under the skin, BC derivatives traced with Krox20 delaminate from nerves and attached to vascular plexus to become mural cells. This “glial vs. vascular” switch occurs between E12.5 and E13.5 and is massive since in the new born skin 30% of pericytes originate from BCs. Transcriptomic comparison of traced cells before and after detachment from nerves led us to identify candidates potentially involved in this mechanism. Among them, Robo3 and its interactors appeared particularly interesting since their expression is restricted to glial cells just before they delaminate from nerves pointing to a role in the detachment. Finally, single cell transcriptomic analysis of BCs before their emigration to the periphery led us to identify clusters of cells with distinct molecular signature supporting their early specification into glial or mural fate. Altogether, our findings suggest a new model for the vasculature development in which glial cells provide first a cocktail of diffusible molecules to shape the vascular plexus into primitive blood vessels, before acting as a major source of mural cells to ensure their maturation into functional blood vessels.

Off-Target Deletion of Conditional Dbc1 Allele in the Foxp3YFP-Cre Mouse Line under Specific Setting.

The Cre-LoxP conditional knockout strategy has been used extensively to study gene function in a specific cell-type. In this study, the authors tried to engineer mice in which the Dbc1 gene is conditionally knocked out in Treg cells. Unexpectedly, the conditional Dbc1 allele was completely deleted with a low frequency in some Foxp3YFP-Cre mice harboring floxed Dbc1 allele under specific settings. It was found that the germline recombination of floxed Dbc1 allele, which caused Dbc1 knock out mice, occurred in the male Foxp3YFP-Cre mice harboring floxed Dbc1 allele. Even though the authors documented that Foxp3 is expressed in the testis, the germline recombination was not caused by the germline expression of Cre, which was driven by the Foxp3 promoter. The germline recombination may be caused by the unspecific expression of Cre recombinase in the fetus, in which the floxed Dbc1 allele of some stem cells with development potential to germ cells may be recombined. Additionally, this study found that the floxed Dbc1 allele was recombined in non-T cells of some Foxp3Cre Dbc1fl mice, which need to be characterized. Our results also suggest that using male mice with a low frequency of recombined gene allele can reduce the risk of having full knock out mice.

High-Throughput Mapping of Long-Range Neuronal Projection Using In Situ Sequencing

Understanding neural circuits requires deciphering interactionsamong myriad cell types defined by spatial organization, connectivity, gene expression, and other properties. Resolving these cell types requires both single-neuron resolution and high throughput, a challenging combination with conventional methods. Here, we introduce barcoded anatomy resolved by sequencing (BARseq), a multiplexed method based on RNA barcoding for mapping projections of thousands of spatially resolved neurons in a single brain and relating those projections to other properties such as gene or Cre expression. Mapping the projections to 11 areas of 3,579 neurons in mouse auditory cortex using BARseq confirmed the laminar organization of the three top classes (intratelencephalic [IT], pyramidal tract-like [PT-like], and corticothalamic [CT]) of projection neurons. In depth analysis uncovered a projection type restricted almost exclusively to transcriptionally defined subtypes of IT neurons. By bridging anatomical and transcriptomic approaches at cellular resolution with high throughput, BARseq can potentially uncover the organizing principles underlying the structure and formation of neural circuits.

Vdr expression in osteoclast precursors is not critical in bone homeostasis

The long-recognized role of the vitamin D endocrine system is to maintain stable serum calcium concentrations, which are ensured by a complex interplay between parathyroid gland, kidney, intestine, and bone. However, although VDR is expressed in osteoclastogenic cells, the contribution of VDR-mediated signaling to osteoclast differentiation and activity remains undefined. We therefore deleted Vdr expression efficiently and specifically in myeloid cells by use of M lysozyme-driven Cre expression, which targets granulocytes, monocytes, macrophages and osteoclasts (Vdrmyel− mice). Bone and calcium homeostasis were investigated under basal conditions and in conditions of increased bone remodeling, by feeding Vdrmyel− and Vdrmyel+ (wildtype) mice either a normal (1%) or a low (0.02%) calcium diet from weaning onwards. Vdrmyel− mice developed normally and were normocalcemic at the age of 8 weeks, both at the normal and the low calcium diet. No differences in trabecular or cortical bone mass were observed between Vdrmyel− mice and their wildtype littermates. Dietary calcium restriction resulted in a comparable reduction of trabecular bone mass (40%) and cortical thickness (48%) in Vdrmyel− and Vdrmyel+ mice, pointing to a massive transfer of calcium from the bone to the serum. In agreement with these results, osteoclastic differentiation of hematopoietic cells of Vdrmyel− mice, either induced by M-CSF and RANKL, or cocultured with osteoblasts, occurred as efficiently as osteoclastogenesis from Vdrmyel+ mice. In conclusion, our data do not support a role for osteoclastic Vdr signaling in the control of bone homeostasis.

Targeting Adeno-Associated Virus Vectors for Local Delivery to Fractures and Systemic Delivery to the Skeleton

A panel of 18 recombinant adeno-associated virus (rAAV) variants, both natural and engineered, constitutively expressing Cre recombinase under the cytomegalovirus early enhancer/chicken β actin (CAG) promoter, were screened for their ability to transduce bone in Ai9 fluorescent reporter mice. Transgenic Cre-induced tdTomato expression served as a measure of transduction efficiency and alkaline phosphatase (AP) activity as an osteoblastic marker. Single injections of AAV8, AAV9, and AAV-DJ into midshaft tibial fractures yielded robust tdTomato expression in the callus. Next, the bone cell-specific promoters Sp7 and Col2.3 were tested to restrict Cre expression in an alternate model of systemic delivery by intravenous injection. Although CAG promoter constructs packaged into AAV8 produced high levels of tdTomato in the bone, liver, heart, spleen, and kidney, bone-specific promoter constructs restricted Cre expression to osseous tissues. AAV variants were further tested in vitro in a human osteoblast cell line (hFOB1.19), measuring GFP reporter expression by flow cytometry after 72 h. AAV2, AAV5, and AAV-DJ showed the highest transduction efficiency. In summary, we produced AAV vectors for selective and high-efficiency in vivo gene delivery to murine bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion postnatally in floxed mouse models.

Knock-In Rat Lines with Cre Recombinase at the Dopamine D1 and Adenosine 2a Receptor Loci.

Genetically modified mice have become standard tools in neuroscience research. Our understanding of the basal ganglia in particular has been greatly assisted by BAC mutants with selective transgene expression in striatal neurons forming the direct or indirect pathways. However, for more sophisticated behavioral tasks and larger intracranial implants, rat models are preferred. Furthermore, BAC lines can show variable expression patterns depending upon genomic insertion site. We therefore used CRISPR/Cas9 to generate two novel knock-in rat lines specifically encoding Cre recombinase immediately after the dopamine D1 receptor (Drd1a) or adenosine 2a receptor (Adora2a) loci. Here, we validate these lines using in situ hybridization and viral vector mediated transfection to demonstrate selective, functional Cre expression in the striatal direct and indirect pathways, respectively. We used whole-genome sequencing to confirm the lack of off-target effects and established that both rat lines have normal locomotor activity and learning in simple instrumental and Pavlovian tasks. We expect these new D1-Cre and A2a-Cre rat lines will be widely used to study both normal brain functions and neurological and psychiatric pathophysiology.

Characterization of a BAC transgenic mouse expressing Krt19-driven iCre recombinase in its digestive organs.

Cytokeratin 19 (KRT19) protein is highly expressed in the epithelium of the gastrointestinal (GI) tract, hepatobiliary tissues, and pancreas of humans and mice. In the present study, we used an improved Cre (iCre) gene to enhance the efficiency of Cre expression in mammalian cells. We established a new transgenic Krt19-iCre bacterial artificial chromosome (BAC) mouse model using the BAC recombineering strategy. Site-specific iCre expression pattern was examined in embryos, adults, and elderly Krt19-iCre mice crossed with Tomato or LacZ reporter mice. Both iCre and reporter protein expressions in adult Krt19-iCre;Tomatoflox/+ (Krt19-iCre Tomato reporter) mice were observed mainly in the epithelial cells of the GI tract, hepatobiliary tissues, and pancreas. However, the expression in the intrahepatic and small pancreatic duct were lower than those in the common bile and large pancreatic duct. In the Krt19-iCre; LacZ reporter embryos, β-galactosidase for the LacZ reporter was expressed in the glandular epithelial cells of the GI tract in 9.5-day embryos, 12-day embryos, and newborn mice. The reporter protein expression in Krt19-iCre-Tomato reporter mice was consistent with the KRT19 expression in human GI tissues. In conclusion, Krt19-iCre BAC transgenic mice can be used to investigate developmental and pathological conditions using the iCre-loxP system.

Development of a Cre-loxP-based genetic system in Aspergillus niger ATCC1015 and its application to construction of efficient organic acid-producing cell factories

The filamentous fungus Aspergillus niger is widely used in the biotechnology industry for the production of chemicals and enzymes. Engineering of this valuable organism to improve its productivity is currently hampered by the lack of efficient genetic tools. Here, a Cre-loxP-based system for gene editing in A. niger was developed and its application in construction of A. niger cell factories to produce various organic acids was explored. Two established inducible systems, the xylanase A gene promoter Pxln and Tet-on system, were examined for driving cre expression and thus selection marker hyh deletion. Under inducing conditions, the efficiency of loxP site-specific recombination in the strain with cre driven by Pxln is about 2%, while cre driven by Tet-on system is about 34% which was used as the platform strain for further genetic engineering. As a proof of application of this system, strains containing different copies of oxaloacetate acetylhydrolase-encoding gene (oahA) were constructed, and the resultant strain S428 showed as high as 3.1-fold increase in oxalic acid production. Furthermore, an efficient malate-producing strain was generated through four-step genetic manipulation (oahA deletion, pyc, mdh3 and C4-dicarboxylate transporter gene c4t318 insertion). The resultant strain S575 achieved a titer 120.38 g/L malic acid with the flask culture, and a titer 201.24 g/L malic acid in fed-batch fermentation. These results demonstrated that this modified Cre-loxP system is a powerful tool for genetic engineering in A. niger, which has the potential to be genetically modified as a viable aciduric platform strain to produce high levels of various organic acids.

Adipocyte-specific disruption of ATPase copper transporting α in mice accelerates lipoatrophy.

ATPase copper transporting α (ATP7A), also known as Menkes disease protein, is a P-type ATPase that transports copper across cell membranes. The critical role of ATP7A-mediated copper homeostasis has been well recognised in various organs, such as the intestine, macrophages and the nervous system. However, the importance of adipocyte ATP7A-mediated copper homeostasis on fat metabolism is not well understood. Here, we sought to reveal the contribution of adipose ATP7A to whole-body fat metabolism in mice. We generated adipocyte-specific Atp7a-knockout (ASKO) mice using the Cre/loxP system, with Cre expression driven by the adiponectin promoter. ASKO mice and littermate control mice were aged on a chow diet or fed with a high-fat diet (HFD); body weight, fat mass, and glucose and insulin metabolism were analysed. Histological analysis, transmission electron microscopy and RNA-sequencing (RNA-Seq) analysis of white adipose tissue (WAT) were used to understand the physiological and molecular changes associated with loss of copper homeostasis in adipocytes. Significantly increased copper concentrations were observed in adipose tissues of ASKO mice compared with control mice. Aged or HFD-fed ASKO mice manifested a lipoatrophic phenotype characterised by a progressive generalised loss of WAT. Dysfunction of adipose tissues in these ASKO mice was confirmed by decreased levels of both serum leptin and adiponectin and increased levels of triacylglycerol and insulin. Systemic metabolism was also impaired in these mice, as evidenced by a pronounced glucose intolerance, insulin resistance and hepatic steatosis. Moreover, we demonstrate a significant induction of lipolysis and DNA-damage signalling pathways in gonadal WAT from aged and HFD-fed ASKO mice. In vitro studies suggest that copper overload is responsible for increased lipolysis and DNA damage. Our results show a previously unappreciated role of adipocyte Atp7a in the regulation of ageing-related metabolic disease and identify new metallophysiologies in whole-body fat metabolism. The datasets generated during the current study are available in the Genome Sequence Archive in BIG Data Center, Beijing Institute of Genomics (BIG), Chinese Academy of Sciences, under accession number CRA001769 (http://bigd.big.ac.cn/gsa).

Targeting adeno-associated viral vectors to fractures and the skeleton

Background & Aim Gene therapy in the context of the bone remains an underdeveloped field, and most approaches have focused on local delivery of therapeutic transgenes. Generating delivery systems capable of affecting the entire skeleton is particularly challenging compared to discrete organs. Viral vectors have great potential as delivery systems for gene editing technology. In particular, adeno-associated viruses (AAVs) have been identified as ideal candidates for gene therapy due to their high efficiency, low immunogenicity, and selective tropism towards different tissues. In this study we aimed to assess a panel of natural and engineered AAV variants for their tropism towards murine bone cells. Methods, Results & Conclusion We screened 18 natural and engineered recombinant AAV (rAAV) variants. AAVs constitutively expressed Cre-recombinase (under a CAG promoter) and transduction efficiency was assessed using a local injection into a murine tibial fracture model using the Ai9 tdTomato Cre reporter mouse line. Fluorescent alkaline phosphatase (AP) staining was used to identify whether transduced cells were osteoblastic. A single injection of rAAV was administered into midshaft tibial fractures at surgery and yielded robust tdTomato expression within the callus at 2 weeks for AAV8, AAV9, and AAV-DJ (Fig. 1a). Next, a model of systemic delivery by intravenous injection into the tail vein of Ai9 mice was performed, with Cre-expression restricted using bone cell-specific promoters Sp7 and Col2.3. At moderate doses (5 × 1011/mouse or ∼2.5 × 1013/kg) tdTomato+ osteoblasts and osteocytes were observed throughout the long bones (Fig. 1b). Minimal tdTomato expression was observed in other tissues including brain, heart, lung, liver, and spleen, indicating both Sp7 and Col2.3 were able to largely restrict Cre expression to bone cells. To summarize, we have identified AAV variants with a high tropism for murine bone cells, and vectors for high efficiency in vivo gene delivery to bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion in floxed mouse models in post-natal bone. Future work will validate AAV vectors that enable targeted CRISPR gene editing that have relevance to the treatment of genetic bone disease.

Generation of Amelx-iCre Mice Supports Ameloblast-Specific Role for Stim1

The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre (iCre)–recombinase from the locus of the mouse ameloblast-specific gene amelogenin X (Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding β-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific β-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 (Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice (Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14-Cre/Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.

Proximal Lck Promoter-Driven Cre Function Is Limited in Neonatal and Ineffective in Adult γδ T Cell Development.

During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in αβ and γδ T cells. We found that pLckCre failed to efficiently delete floxed genes in γδ T cells in contrast to a complete deletion in conventional as well as unconventional αβ T cells. Mechanistically, γδ T cells inefficiently transcribed the endogenous proximal Lck promoter compared with αβ T cells during adult thymic development. A small population of γδ T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17- or IFN-γ-producing γδ T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of γδ T cells with recombined floxed genes that were increased in unique γδ T subsets, such as the IL-17-producing γδ T cells. Our data point out striking differences in Lck transcription between perinatal and adult γδ T cell development. Taken together, the data presented in this study shed new light on γδ T cell development and stimulate a reanalysis of data generated using the pLckCre transgenic mice.

Conditional loss of ERK1 and ERK2 results in abnormal placentation and delayed parturition in the mouse

Extracellular-signal-regulated kinases (ERK) 1 and 2 regulate many aspects of the hypothalamic-pituitary-gonadal axis. We sought to understand the role of ERK1/2 signaling in cells expressing a Cre allele regulated by the endogenous GnRHR promoter (GRIC-ERKdko). Adult female GRIC-ERKdko mice were hypogonadotropic and anovulatory. Gonadotropin administration and mating led to pregnancy in one-third of the ERKdko females. Litters from ERKdko females and pup weights were reduced coincident with delayed parturition and 100% neonatal mortality. Based on this, we examined Cre expression in implantation sites as a potential mechanism. GnRHR mRNA levels at e10.5 and e12.5 were comparable to pituitary levels from adult female mice at proestrus and GnRHR mRNA in decidua was enriched compared to whole implantation site. In vivo studies confirmed recombination in decidua, and GRIC-ERKdko placentas showed reduced ERK2 expression. Histopathology revealed abnormalities in placental architecture in the GRIC-ERKdko animals. Regions of apoptosis at the decidual/uterine interface at e18.5 were observed in control animals but apoptotic tone in these regions was reduced in ERKdko animals. These studies support a potential model of ERK-dependent signaling within the implantation site leading to loss of placental architecture and mis-regulation of apoptotic events at parturition occurring coincident with prolonged gestation and neonatal mortality.

Abstract 3459: PKD1 regulates susceptibility to ulcerative colitis and colorectal cancer

Abstract Compromised integrity of the epithelial barrier function of colon tissue associated with inflammatory bowel diseases (IBDs) or arising from tumor-elicited inflammation (TEI) are associated with and support formation of colorectal cancer (CRC). Apical tight junction (TJ) proteins, including multiple specific claudins, are critical in regulating TJ barrier function and paracellular permeability. Changes in TJ composition in the kidney are strongly linked to the etiology of autosomal dominant polycystic kidney disease (ADPKD), with ADPKD-inducing mutations in PKD1 causing non-leaky barriers that can withstand high hydrostatic pressure within renal cysts a hallmark of this common (1 in 500) inherited disease. Intriguingly, a large population study has found a decreased incidence of CRC in ADPKD patients. Based on these and other suggestive data, we hypothesized that loss of PKD1 would hinder development of ulcerative colitis and initiation of colitis associated cancer, based on reorganizing TJs and TEI in the colon. To evaluate the impact of Pkd1 loss on colon barrier function, we treated 10-12 week old wt or Pkd1fl/fl mice with tamoxifen-induced, Cre expression from the CreERT2 cassette for 5 days with 2.5% DSS in drinking water to induce acute colitis, compared to a no-DSS control group. For the wtcohort, treatment with DSS increased orally gavaged FITC-dextran detectable in serum versus controls. In contrast, no Pkd1-/- mice showed a similar DSS-dependent response to FITC-dextran, suggesting reinforced colon barrier function; further, histopathological assessment confirmed less DSS-induced damage in Pkd1fl/fl mice. Further, based on immunofluorescence (IF) analysis of tissue sections, claudins 4 and 7 associated with increased barrier function) strongly elevated in the colonic epithelium of Pkd1fl/fl versus wt mice, with the expressed proteins having consistently greater localization to cell junctions in Pkd1fl/fl versus wt mice. We also investigated the formation of CRC tumors, using a CDX2-ERT2/Cre model for tamoxifen-induced loss of Apc and/or Pkd1 in the colon to compare tumorigenesis in the Apcfl/fl, Apcfl/fl Pkd1fl/fl, and Apcfl/flPkd1fl/+ genotypes. We observed a highly significant progressive increase in the number and size of Apc mutation-induced tumors based on retention of the Pkd1 gene. Preliminary qRT-PCR analysis of these tumors shows that a Pkd1fl/fl genotype substantially increases CLDN4 in normal and tumor tissue, and decreases TNFα expression in tumors, suggesting reduced inflammation. This work and extended mechanistic characterization identifies PKD1 as a vital regulator of signaling systems already associated with colitis and CRC. Understanding the role of PKD1 and its effectors may provide useful information to genetic counselors assessing risk of IBD and CRC, and suggest therapeutic strategies. Citation Format: Anna S. Nikonova, Anna Kiseleva, Ilya Serebriiskii, Sergei Grivennikov, Erica A. Golemis. PKD1 regulates susceptibility to ulcerative colitis and colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3459.

Abstract 1604: Modulating effects of genistein in a mouse model of conditional BRCA1 deletion and triple negative breast cancer cells

Abstract Germline mutations in BRCA1 (BRCA1+/-) confer ~72% risk of breast cancer, the majority of which are triple negative breast cancer (TNBC). Hypermethylation of BRCA1 is observed at high frequency in sporadic TNBC and may play a role in tumor development in BRCA1 mutation carriers. The aryl hydrocarbon receptor (AhR) coordinates epigenetic silencing of BRCA1 and is overexpressed in some TNBC. The purpose of our study is to investigate the effect of dietary genistein on BRCA1 epigenetic regulation, the AhR pathway and mammary tumorigenesis in BRCA1+/- and BRCA1+/+ mice. We used Cre-lox recombination to derive a mouse model of conditional BRCA1+/-. We used BRCA1F22/24 (The Jackson Laboratory, stock no. 017835) mice, which harbor loxP sites flanking exons 22-24 of the BRCA1 gene. We crossed dams that were heterozygous for BRCA1F22/24 (BRCA1+/FL) and transgenic for Cre recombinase (TG) under control of the mouse mammary tumor virus (MMTV) promoter with wildtype NJ males to derive our colony. Breeding pairs were administered either control or genistein-enriched (4 ppm) diets upon set up, which were continued throughout the life of the offspring. Changes in mRNA expression and protein levels were determined, respectively, by RT-PCR and Western blot. Crewas not expressed in mammary glands of non-TG offspring and Cre expression had no effect on BRCA1 mRNA levels in BRCA1+/+ mice, which do not harbor BRCA1 loxP sites. BRCA1 expression was ~40% decreased in mammary glands of TG, BRCA1+/FL mice compared with TG, BRCA1+/+ mice. Hepatic BRCA1 expression was not changed, confirming conditional BRCA1 knockdown. Preliminary data indicate BRCA1 expression in mammary glands of genistein-fed TG, BRCA1+/+ mice was elevated (~1.3 fold) compared with control-fed mice. Moreover, the genistein diet induced a compensatory increase in BRCA1 expression in mammary glands of TG, BRCA1+/FL mice. We found genistein rescued BRCA1 levels in TNBC HCC38 cells that have constitutively active AhR and hypermethylated BRCA1. These data suggest dietary genistein may have protective effects against tumorigenesis in these mice. To that end, we are currently monitoring tumor development, BRCA1 methylation, and changes in the AhR pathway in BRCA1+/+ and BRCA1+/FL mice fed either the genistein-enriched or control diet. Citation Format: Micah G. Donovan, Ornella I. Selmin, Thomas C. Doetschman, Donato F. Romagnolo. Modulating effects of genistein in a mouse model of conditional BRCA1 deletion and triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1604.

Abstract 2733: Novel mouse model carrying APC and PIK3CA mutations in colorectal cancer

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in the United States, despite substantial development in its early diagnosis and treatment. The animal models of CRC have been immensely useful for understanding CRC pathogenesis, investigating the effects of genetic modifications on CRC, and for the development of new chemopreventive/chemotherapeutic drugs. Most (>80%) CRC carry mutations in the APC gene and many (15-20%) carry mutations in the PIK3CA gene, encoding the p110 catalytic subunit of the PI3K kinase. We sought to better understand the interaction between APC and PIK3CA mutations in the mammalian intestine. To examine the effect of mutations in APC and PIK3CA on tumorigenesis, Min mice were crossed with the FC13K1 (FC13K1ApcMin/+). This cross resulted in a murine model with the loss of one allele of Apc throughout the intestine and the expression of a dominant active PI3K (3K1) in the distal small intestine and colon due to the expression of Cre under the control of the rat fatty acid binding protein-1 promoter (FC1). There was increased tumor multiplicity, size and a more aggressive and poorly differentiated phenotype as a consequence of synergy between APC and PIK3CA mutations. Tumors form as adenomas, but quickly progress to invasive adenocarcinomas that eventually metastasize to regional lymph nodes. Using this mouse model, we have recently shown that fisetin, a dietary flavonoid could be used as a preventive agent and an adjuvant with 5-fluorouracil (FU) for the treatment of PIK3CA-mutant CRC. Tumor incidence was markedly lower in fisetin-treated FC13K1ApcMin/+ mice in distal small intestine and colon, as compared to control animals, indicating that fisetin is a strong preventive agent. In addition, the combination of fisetin and 5-FU also reduced the total number of intestinal tumors. We extended our work by demonstrating that the effect of drugs on tumorigenesis was impacted by the mutation profile of the tumor and intratumoral heterogeneity. We developed the mouse model in which intestinal tumors were composed entirely of PIK3CA wild-type cells, entirely of PIK3CA-mutant cells, or a mixture of both. We demonstrated that low dose aspirin blocked the development of heterogeneous tumors composed of PIK3CA wild-type and PIK3CA-mutant cells but not the development of homogenous tumors composed entirely of PIK3CA wild-type cells. Thus, this new model of CRC recapitulates the effect of aspirin that was observed in humans. Sustained exposure to low dose aspirin reduced the recurrence the PIK3CA-mutant CRC cancers in humans. This aggressive murine model is an exciting model of human CRC that has the potential to be instrumental in the development of targeted chemoprevention and therapeutics. Citation Format: Naghma Khan, Farah Jajeh, Emily L. Eberhardt, Devon D. Miller, Dawn M. Albrecht, Rachel Van Doorn, Melissa D. Hruby, Morgan E. Maresh, Linda Clipson, Hasan Mukhtar, Richard B. Halberg. Novel mouse model carrying APC and PIK3CA mutations in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2733.

Abstract 2649: A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells

Abstract Hypoxia occurs in 90% of solid tumors due to inefficient vasculature and has been demonstrated to promote a more aggressive phenotype, correlating with worse patient prognosis. Current methods to measure hypoxia in human tumors only identify cells that are hypoxic at the time the tissue is collected and do not delineate cells that were transiently exposed to hypoxia but survived and adapted. To overcome this challenge, we generated a Lox-Cre dual-vector oxygen-dependent reporter system. Under hypoxia, Cre expression leads to the cleavage of a DsRed reporter and permanent expression of a GFP reporter. We stably transduced breast cancer cell lines and demonstrated an irreversible reporter-switch that occurs when cells are exposed to 0.5% O2, the typical O2 level in human breast tumors, but does not occur under physiological conditions (8% O2). To explore the utility of our system, we embedded spheroids derived from MDA-MB-231 reporter cells in collagen and cultured them under 20% O2 conditions. A defined hypoxic core (GFP+) was visible after 15 days in culture and it correlated with hypoxic O2 measurements. We then generated orthotopic breast tumors by transplanting MDA-MB-231 reporter cells into the mammary fat pad of mice. Hypoxic cells (GFP+) were localized in the peri-necrotic region of the tumor and co-localized with hypoxyprobe and HIF1α labeling. RNA sequencing of GFP+ compared to DsRed+ tumor cells demonstrated that GFP+ cells were enriched for the hallmark signature of hypoxia. Additionally, we developed a transgenic mouse expressing the hypoxia-driven construct that was bred to a tdTomato-floxed GFP mouse. A female double fluorescent/hypoxia-reporter mouse was then bred to a male FVB/N-Tg(MMTV-PyMT) mouse in order to generate triple-transgenic mice that form breast tumors that spontaneously metastasize. We systematically studied hypoxia in spontaneous breast tumors from the development of ductal hyperplasia to late stage invasive carcinoma. Tumor hypoxia was first detected in early stage ductal carcinoma in situ (DCIS) lesions. Similarly to the spheroid model, mammary organoids derived from triple-transgenic mice tumors cultured under 20% O2 conditions also exhibited a defined hypoxic core after 15 days in culture. Both orthotopic and transgenic mouse models revealed an increased potency of post-hypoxic cells at arriving to distant sites in early stage metastasis. We utilized RNA sequencing to identify potential mechanisms of enhanced metastatic capacity. Ultimately, our system allowed the fate-map of hypoxic cells during tumor progression to metastasis. We show for the first time that 3D in vitro organoids derived from PyMT tumors develop regions of hypoxia under normal tissue culture conditions. In vivo, we show that hypoxic cells are more potent at arriving at distant sites and that they retain a different gene expression that potentially confers a metastatic advantage. Citation Format: Inês Godet, Yu Jung Shin, Julia A. Ju, Soumitra Bhoyar, I Chae Ye, Guannan Wang, Saraswati Sukumar, Daniele M. Gilkes. A novel approach to fate-map hypoxic cells during tumor progression uncovers metastatic potency of post-hypoxic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2649.

A new mouse model to study the role of ectopic Nanos3 expression in cancer

BackgroundNANOS3 is a gene conserved throughout evolution. Despite the quite low conservation of Nanos sequences between different organisms and even between Nanos paralogs, their role in germ cell development is remarkably universal. Human Nanos3 expression is normally restricted to the gonads and the brain. However, ectopic activation of this gene has been detected in various human cancers. Until now, Nanos3 and other Nanos proteins have been studied almost exclusively in germ cell development.MethodsTransgenic mice were generated by targeted insertion of a human Nanos3 cDNA into the ROSA26 locus. The transgene could be spatiotemporally induced by Cre recombinase activity removing an upstream floxed STOP cassette. A lung tumor model with ectopic Nanos3 expression was based on the lung-specific activation of the reverse tetracycline transactivator gene, in combination with a tetO-CMV promoter controlling Cre expression. When doxycycline was provided to the mice, Cre was activated leading to deletion of TP53 alleles and activation of both oncogenic KRasG12D and Nanos3. Appropriate controls were foreseen. Tumors and tumor-derived cell cultures were analyzed in various ways.ResultsWe describe the successful generation of Nanos3LSL/− and Nanos3LSL/LSL mice in which an exogenous human NANOS3 gene can be activated in vivo upon Cre expression. These mice, in combination with different conditional and doxycycline-inducible Cre lines, allow the study of the role of ectopic Nanos3 expression in several cancer types. The Nanos3LSL mice were crossed with a non-small cell lung cancer (NSCLC) mouse model based on conditional expression of oncogenic KRas and homozygous loss of p53. This experiment demonstrated that ectopic expression of Nanos3 in the lungs has a significant negative effect on survival. Enhanced bronchiolar dysplasia was observed when Nanos3-expressing NSCLC mice were compared with control NSCLC mice. An allograft experiment, performed with cell cultures derived from primary lung tumors of control and Nanos3-expressing NSCLC mice, revealed lymph node metastasis in mice injected with Nanos3-expressing NSCLC cells.ConclusionsA new mouse model was generated allowing examination of Nanos3-associated pathways and investigation of the influence of ectopic Nanos3 expression in various cancer types. This model might identify Nanos3 as an interesting target in cancer therapeutics.

Modeling Breast Cancer via an Intraductal Injection of Cre-expressing Adenovirus into the Mouse Mammary Gland.

Breast cancer is a heterogeneous disease, possibly due to complex interactions between different cells of origins and oncogenic events. Mouse models are instrumental in gaining insights into these complex processes. Although many mouse models have been developed to study contributions of various oncogenic events and cells of origin to breast tumorigenesis, these models are often not cell-type or organ specific or cannot induce the initiation of mammary tumorigenesis in a temporally controlled manner. Here we describe a protocol to generate a new type of breast cancer mouse models based on the intraductal injection of Cre-expressing adenovirus (Ad-Cre) into mouse mammary glands (MGs). Due to the direct injection of Ad-Cre into mammary ducts, this approach is MG specific, without any unwanted cancer induction in other organs. The intraductal injection procedure can be performed in mice at different stages of their MG development (thus, it permits temporal control of cancer induction, starting from ~3-4 weeks of age). The cell-type specificity can be achieved by using different cell-type-specific promoters to drive Cre expression in the adenoviral vector. We show that luminal and basal mammary epithelial cells (MECs) can be tightly targeted for Cre/loxP-based genetic manipulation via an intraductal injection of Ad-Cre under the control of the Keratin 8 or Keratin 5 promoter, respectively. By incorporating a conditional Cre reporter (e.g., Cre/loxP-inducible Rosa26-YFP reporter), we show that MECs targeted by Ad-Cre, and tumor cells derived from them, can be traced by following the reporter-positive cells after intraductal injection.