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https://www.readbyqxmd.com/read/29336844/detecting-and-avoiding-problems-when-using-the-cre-lox-system
#1
REVIEW
Allisa J Song, Richard D Palmiter
The Cre-lox recombination approach is commonly used to generate cell-specific gene inactivation (or activation). We have noticed that the breeding and genotyping sections of papers utilizing Cre-lox techniques are frequently incomplete. While seemingly straightforward, there are important considerations that need to be implemented in the breeding and genotyping methods to prevent the introduction of experimental confounds. Germline recombination and transient expression of Cre recombinase during development are some examples of the complications that can occur, and conventional genotyping methods may fail to identify these events...
January 11, 2018: Trends in Genetics: TIG
https://www.readbyqxmd.com/read/29324782/mafb-is-dispensable-for-the-fetal-testis-morphogenesis-and-the-maintenance-of-spermatogenesis-in-adult-mice
#2
Hossam H Shawki, Hisashi Oishi, Toshiaki Usui, Yu Kitadate, Walaa A Basha, Ahmed M Abdellatif, Kazunori Hasegawa, Risa Okada, Keiji Mochida, Hany A El-Shemy, Masafumi Muratani, Atsuo Ogura, Shosei Yoshida, Satoru Takahashi
The transcription factor MAFB is an important regulator of the development and differentiation of various organs and tissues. Previous studies have shown that MAFB is expressed in embryonic and adult mouse testes and is expected to act as the downstream target of retinoic acid (RA) to initiate spermatogenesis. However, its exact localization and function remain unclear. Here, we localized MAFB expression in embryonic and adult testes and analyzed its gene function using Mafb-deficient mice. We found that MAFB and c-MAF are the only large MAF transcription factors expressed in testes, while MAFA and NRL are not...
2018: PloS One
https://www.readbyqxmd.com/read/29322407/modelling-intestinal-carcinogenesis-using-in-vitro-organoid-cultures
#3
Thierry Jardé, Genevieve Kerr, Reyhan Akhtar, Helen E Abud
Mouse models of intestinal carcinogenesis are very powerful for studying the impact of specific mutations on tumour initiation and progression. Mutations can be studied both singularly and in combination using conditional alleles that can be induced in a temporal manner. The steps in intestinal carcinogenesis are complex and can be challenging to image in live animals at a cellular level. The ability to culture intestinal epithelial tissue in three-dimensional organoids in vitro provides an accessible system that can be genetically manipulated and easily visualised to assess specific biological impacts in living tissue...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29315333/cell-penetrating-peptide-driven-cre-recombination-in-porcine-primary-cells-and-generation-of-marker-free-pigs
#4
Qianqian Kang, Zhaolin Sun, Zhiyuan Zou, Ming Wang, Qiuyan Li, Xiaoxiang Hu, Ning Li
Cell-penetrating peptides (CPPs) have been increasingly used to deliver various molecules, both in vitro and in vivo. However, there are no reports of CPPs being used in porcine fetal fibroblasts (PFFs). The increased use of transgenic pigs for basic research and biomedical applications depends on the availability of technologies for efficient genetic-modification of PFFs. Here, we report that three CPPs (CPP5, TAT, and R9) can efficiently deliver active Cre recombinase protein into PFFs via an energy-dependent endocytosis pathway...
2018: PloS One
https://www.readbyqxmd.com/read/29314464/chemogenetic-inhibition-of-direct-pathway-striatal-neurons-normalizes-pathological-cue-induced-reinstatement-of-drug-seeking-in-rats
#5
Lindsay M Yager, Aaron F Garcia, Elizabeth A Donckels, Susan M Ferguson
Addiction to drugs such as cocaine is marked by cycles of compulsive drug-taking and drug-seeking behavior. Although the transition to addiction is thought to recruit neural processes in dorsal striatum, little is known regarding the role of dorsal striatal projections to the substantia nigra (i.e. the direct pathway) in regulating these behaviors. Combining a Cre-recombinase-dependent chemogenetic approach with a cocaine self-administration paradigm that produces both low-risk and high-risk addiction phenotypes, we examined the effect of transiently decreasing direct pathway activity in the dorsomedial striatum on drug-taking and drug-seeking under conditions of normal and pathological drug use...
January 5, 2018: Addiction Biology
https://www.readbyqxmd.com/read/29305935/hedgehog-yap-signaling-pathway-regulates-glutaminolysis-to-control-hepatic-stellate-cell-activation
#6
Kuo Du, Jeongeun Hyun, Richard T Premont, Steve S Choi, Gregory A Michelotti, Marzena Swiderska-Syn, George D Dalton, Eric Thelen, Bahar Salimian Rizi, Youngmi Jung, Anna Mae Diehl
BACKGROUND & AIMS: Cirrhosis results from accumulation of myofibroblasts derived from quiescent hepatic stellate cells (Q-HSCs); it regresses when myofibroblastic HSCs are depleted. Hedgehog signaling promotes transdifferentiation of HSCs by activating Yes-associated protein 1 (YAP1 or YAP) and inducing aerobic glycolysis. However, increased aerobic glycolysis alone cannot meet the high metabolic demands of myofibroblastic HSCs. Determining the metabolic processes of these cells could lead to strategies to prevent progressive liver fibrosis, so we investigated whether glutaminolysis (conversion of glutamine to alpha-ketoglutarate) sustains energy metabolism and permits anabolism when Q-HSCs become myofibroblastic, and whether this is controlled by hedgehog signaling to YAP...
January 3, 2018: Gastroenterology
https://www.readbyqxmd.com/read/29302202/aav-mediated-astrocyte-specific-gene-expression-under-human-aldh1l1-promoter-in-mouse-thalamus
#7
Wuhyun Koh, Yongmin Mason Park, Seung Eun Lee, C Justin Lee
Adeno-associated virus (AAV)-mediated gene delivery has been proposed to be an essential tool of gene therapy for various brain diseases. Among several cell types in the brain, astrocyte has become a promising therapeutic target for brain diseases, as more and more contribution of astrocytes in pathophysiology has been revealed. Until now, genetically targeting astrocytes has been possible by utilizing the glial fibrillary acidic protein (GFAP) promoter. In some brain areas including thalamus, however, the GFAP expression in astrocytes is reported to be low, making it difficult to genetically target astrocytes using GFAP promoter...
December 2017: Experimental Neurobiology
https://www.readbyqxmd.com/read/29300388/colonoscopy-based-colorectal-cancer-modeling-in-mice-with-crispr-cas9-genome-editing-and-organoid-transplantation
#8
Jatin Roper, Tuomas Tammela, Adam Akkad, Mohammad Almeqdadi, Sebastian B Santos, Tyler Jacks, Ömer H Yilmaz
Most genetically engineered mouse models (GEMMs) of colorectal cancer are limited by tumor formation in the small intestine, a high tumor burden that limits metastasis, and the need to generate and cross mutant mice. Cell line or organoid transplantation models generally produce tumors in ectopic locations-such as the subcutaneous space, kidney capsule, or cecal wall-that do not reflect the native stromal environment of the colon mucosa. Here, we describe detailed protocols to rapidly and efficiently induce site-directed tumors in the distal colon of mice that are based on colonoscopy-guided mucosal injection...
February 2018: Nature Protocols
https://www.readbyqxmd.com/read/29290101/the-competence-system-of-streptococcus-anginosus-and-its-use-for-genetic-engineering
#9
Richard Bauer, Stefanie Mauerer, Aline Grempels, Barbara Spellerberg
Streptococcus anginosus is considered a human commensal but improvements in species identification in recent years highlighted its role as an emerging pathogen. However, our knowledge about the pathogenicity mechanisms in this species is rare. One reason for this is the lack of published genetic manipulation techniques in the Streptococcus anginosus group. To establish a novel mutation technique we investigated the competence system of S. anginosus and created a Cre-recombinase based mutation method that allows the generation of markerless gene deletions in S...
December 31, 2017: Molecular Oral Microbiology
https://www.readbyqxmd.com/read/29286375/inducing-cre-lox-recombination-in-mouse-cerebral-cortex-through-in-utero-electroporation
#10
Katherine M Bland, Zachary O Casey, Christopher J Handwerk, Z Logan Holley, George S Vidal
Cell-autonomous neuronal functions of genes can be revealed by causing loss or gain of function of a gene in a small and sparse population of neurons. To do so requires generating a mosaic in which neurons with loss or gain of function of a gene are surrounded by genetically unperturbed tissue. Here, we combine the Cre-lox recombination system with in utero electroporation in order to generate mosaic brain tissue that can be used to study the cell-autonomous function of genes in neurons. DNA constructs (available through repositories), coding for a fluorescent label and Cre recombinase, are introduced into developing cortical neurons containing genes flanked with loxP sites in the brains of mouse embryos using in utero electroporation...
November 17, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/29248441/kr%C3%A3-ppel-like-factor-5-increased-in-pancreatic-ductal-adenocarcinoma-promotes-proliferation-acinar-to-ductal-metaplasia-pancreatic-intraepithelial-neoplasia-and-tumor-growth-in-mice
#11
Ping He, Jong Won Yang, Vincent W Yang, Agnieszka B Bialkowska
BACKGROUND & AIMS: Activating mutations in KRAS are detected in most pancreatic ductal adenocarcinomas (PDACs). Expression of an activated form of KRAS (KrasG12D) in pancreata of mice is sufficient to induce formation of pancreatic intraepithelial neoplasia (PanINs)-a precursor of PDAC. Pancreatitis increases formation of PanINs in mice that express KrasG12D by promoting acinar to ductal metaplasia (ADM). We investigated the role of the transcription factor Krüppel-like factor 5 (KLF5) in ADM and KRAS-mediated formation of PanINs...
December 14, 2017: Gastroenterology
https://www.readbyqxmd.com/read/29244772/sphingosine-1-phosphate-receptor-1-expressed-in-myeloid-cells-slows-diet-induced-atherosclerosis-and-protects-against-macrophage-apoptosis-in-ldlr-ko-mice
#12
Leticia Gonzalez, Alexander S Qian, Usama Tahir, Pei Yu, Bernardo L Trigatti
We generated myeloid specific sphingosine-1-phosphate receptor 1 (S1pr1) deficient mice by crossing mice that had myeloid specific expression of Cre recombinase (lyzMCre) with mice having the S1pr1 gene flanked by loxP recombination sites. We transplanted bone marrow from these mice and control lyzMCre mice with intact macrophage S1pr1 gene expression into low-density lipoprotein (LDL) receptor gene (Ldlr) deficient mice. The resulting chimeras were fed a high fat atherogenic diet for nine or twelve weeks and evaluated for atherosclerosis development in the aortic sinus...
December 15, 2017: International Journal of Molecular Sciences
https://www.readbyqxmd.com/read/29234141/cannabinoid-type-2-receptors-in-dopamine-neurons-inhibits-psychomotor-behaviors-alters-anxiety-depression-and-alcohol-preference
#13
Qing-Rong Liu, Ana Canseco-Alba, Hai-Ying Zhang, Patricia Tagliaferro, Monika Chung, Eugene Dennis, Branden Sanabria, Norman Schanz, Joao Carlos Escosteguy-Neto, Hiroki Ishiguro, Zhicheng Lin, Susan Sgro, Claire M Leonard, Jair Guilherme Santos-Junior, Eliot L Gardner, Josephine M Egan, Jeung Woon Lee, Zheng-Xiong Xi, Emmanuel S Onaivi
Cannabinoid CB2 receptors (CB2Rs) are expressed in mouse brain dopamine (DA) neurons and are involved in several DA-related disorders. However, the cell type-specific mechanisms are unclear since the CB2R gene knockout mice are constitutive gene knockout. Therefore, we generated Cnr2-floxed mice that were crossed with DAT-Cre mice, in which Cre- recombinase expression is under dopamine transporter gene (DAT) promoter control to ablate Cnr2 gene in midbrain DA neurons of DAT-Cnr2 conditional knockout (cKO) mice...
December 12, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29225599/promoter-specificity-and-efficacy-in-conditional-and-inducible-transgenic-targeting-of-lung-macrophages
#14
Alexandra L McCubbrey, Kristen C Allison, Alisa B Lee-Sherick, Claudia V Jakubzick, William J Janssen
Conditional and inducible Cre-loxP systems are used to target gene deletion to specific cell lineages and tissues through promoter-restricted expression of the bacterial DNA recombinase, Cre. Although Cre-loxP systems are widely used to target gene deletion in lung macrophages, limited data are published on the specificity and efficiency of "macrophage targeting" Cre lines. Using R26-stopfl/fl-TdTomato and tetOn-GFP reporter lines, we assessed the specificity and efficiency of four commercially available Cre driver lines that are often considered "macrophage specific...
2017: Frontiers in Immunology
https://www.readbyqxmd.com/read/29222692/advances-in-understanding-hilar-mossy-cells-of-the-dentate-gyrus
#15
REVIEW
Helen E Scharfman
Hilar mossy cells (MCs) of the dentate gyrus (DG) distinguish the DG from other hippocampal subfields (CA1-3) because there are two glutamatergic cell types in the DG rather than one. Thus, in the DG, the main cell types include glutamatergic granule cells (GCs) and MCs, whereas in CA1-3, the only glutamatergic cell type is the pyramidal cell. In contrast to GCs, MCs are different in morphology, intrinsic electrophysiological properties, afferent input and axonal projections, so their function is likely to be very different from GCs...
December 8, 2017: Cell and Tissue Research
https://www.readbyqxmd.com/read/29221447/disruption-of-postnatal-folliculogenesis-and-development-of-ovarian-tumor-in-a-mouse-model-with-aberrant-transforming-growth-factor-beta-signaling
#16
Yang Gao, Xin Fang, David F Vincent, David W Threadgill, Laurent Bartholin, Qinglei Li
BACKGROUND: Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes. METHODS: A mouse model containing constitutively active TGFBR1 (TGFBR1CA) using Gdf9-iCre (termed TGFBR1-CAG9Cre) was generated...
December 8, 2017: Reproductive Biology and Endocrinology: RB&E
https://www.readbyqxmd.com/read/29220094/osteocytes-play-an-important-role-in-experimental-periodontitis-in-healthy-and-diabetic-mice-through-expression-of-rankl
#17
Dana T Graves, Ahmed Alshabab, Mayra Laino Albiero, Marcelo Mattos, Joice Dias Correa, Shanshan Chen, Yang Yang
AIM: Periodontitis results from bacteria induced inflammation. A key cytokine, RANKL is produced by a number of cell types. The cellular source of RANKL critical for periodontitis has not been established. METHODS: We induced periodontal bone loss by oral inoculation of Porphyromonas gingivalis and Fusobacterium nucleatum in both normoglycemic and streptozotocin induced type 1 diabetic mice. Experimental transgenic mice had osteocyte specific deletion of floxed RANKL mediated by DMP-1 driven Cre recombinase...
December 8, 2017: Journal of Clinical Periodontology
https://www.readbyqxmd.com/read/29218825/in-vivo-evidence-supporting-a-metastasis-suppressor-role-for-stard13-dlc2-in-erbb2-neu-oncogene-induced-mouse-mammary-tumours
#18
Pratima Basak, Heather Leslie, Rachelle L Dillon, William J Muller, Afshin Raouf, Michael Ra Mowat
Overexpression of dominant oncogenes and the loss of tumor suppressor genes are basic genetic events in the acquisition of the malignant phenotype. The erb-b2 receptor tyrosine kinase 2 (ERBB-2) proto-oncogene is overexpressed in 20-30% of human breast cancers. The StAR related lipid transfer domain containing 13 gene (STARD13), also known as Deleted in Liver Cancer-2 (DLC-2), maps to chromosome band 13q12.3 and is frequently downregulated in human cancers, including 72% of breast cancers. It encodes a RhoGAP protein with sterile α motif (SAM) and StAR-related lipid transfer (START) domains...
December 8, 2017: Genes, Chromosomes & Cancer
https://www.readbyqxmd.com/read/29217488/incomplete-concordance-of-dopamine-transporter-cre-datirescre-mediated-recombination-and-tyrosine-hydroxylase-immunoreactivity-in-the-mouse-forebrain
#19
Siew Hoong Yip, Jade York, Brian Hyland, Stephen J Bunn, David R Grattan
Co-localization of the expression of the dopamine transporter (DAT) with the catecholamine synthesising enzyme tyrosine hydroxylase (TH) has been investigated using transgenic mice expressing Cre recombinase (Cre) dependent green fluorescent protein (GFP) under the control of the DAT promoter (DATIREScre/GFP). Brain sections from adult female mice were stained for Cre-induced GFP and TH using immunohistochemistry, revealing a high degree of co-expression in the midbrain dopaminergic neurons (A8-10) with the exception of the periaqueductal and dorsal raphe nuclei where dual-labelling was notably lower...
December 4, 2017: Journal of Chemical Neuroanatomy
https://www.readbyqxmd.com/read/29214672/region-specific-deletions-of-the-glutamate-transporter-glt1-differentially-affect-seizure-activity-and-neurodegeneration-in-mice
#20
Junya Sugimoto, Moeko Tanaka, Kaori Sugiyama, Yukiko Ito, Hidenori Aizawa, Miho Soma, Tomoko Shimizu, Akira Mitani, Kohichi Tanaka
Glial glutamate transporter GLT1 plays a key role in the maintenance of extracellular glutamate homeostasis. Recent human genetic studies have suggested that de novo mutations in GLT1 (EAAT2) cause early-onset epilepsy with multiple seizure types. Consistent with these findings, global GLT1 null mice show lethal spontaneous seizures. The consequences of GLT1 dysfunction vary between different brain regions, suggesting that the role of GLT1 dysfunction in epilepsy may also vary with brain regions. In this study, we generated region-specific GLT1 knockout mice by crossing floxed-GLT1 mice with mice that express the Cre recombinase in a particular domain of the ventricular zone...
December 6, 2017: Glia
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