An Inducible and Reversible Mouse Genetic Rescue System

Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications

Essential Role of Chromatin Remodeling Protein Bptf in Early Mouse Embryos and Embryonic Stem Cells

We have characterized the biological functions of the chromatin remodeling protein Bptf (Bromodomain PHD-finger Transcription Factor), the largest subunit of NURF (Nucleosome Remodeling Factor) in a mammal. Bptf mutants manifest growth defects at the post-implantation stage and are reabsorbed by E8.5. Histological analyses of lineage markers show that Bptf−/− embryos implant but fail to establish a functional distal visceral endoderm. Microarray analysis at early stages of differentiation has identified Bptf-dependent gene targets including homeobox transcriptions factors and genes essential for the development of ectoderm, mesoderm, and both definitive and visceral endoderm. Differentiation of Bptf−/− embryonic stem cell lines into embryoid bodies revealed its requirement for development of mesoderm, endoderm, and ectoderm tissue lineages, and uncovered many genes whose activation or repression are Bptf-dependent. We also provide functional and physical links between the Bptf-containing NURF complex and the Smad transcription factors. These results suggest that Bptf may co-regulate some gene targets of this pathway, which is essential for establishment of the visceral endoderm. We conclude that Bptf likely regulates genes and signaling pathways essential for the development of key tissues of the early mouse embryo

Selective Removal of the Selenocysteine tRNA\u3csup\u3e [Ser]Sec\u3c/sup\u3e Gene (\u3ci\u3eTrsp\u3c/i\u3e) in Mouse Mammary Epithelium

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA [Ser]Sec gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA [Ser]Sec amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health

Selective Removal of the Selenocysteine tRNA([Ser]Sec) Gene (Trsp) in Mouse Mammary Epithelium

Mice homozygous for an allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene (Trsp) flanked by loxP sites were generated. Cre recombinase-dependent removal of Trsp in these mice was lethal to embryos. To investigate the role of Trsp in mouse mammary epithelium, we deleted this gene by using transgenic mice carrying the Cre recombinase gene under control of the mouse mammary tumor virus (MMTV) long terminal repeat or the whey acidic protein promoter. While both promoters target Cre gene expression to mammary epithelium, MMTV-Cre is also expressed in spleen and skin. Sec tRNA([Ser]Sec) amounts were reduced by more than 70% in mammary tissue with either transgene, while in skin and spleen, levels were reduced only with MMTV-Cre. The selenoprotein population was selectively affected with MMTV-Cre in breast and skin but not in the control tissue, kidney. Moreover, within affected tissues, expression of specific selenoproteins was regulated differently and often in a contrasting manner, with levels of Sep15 and the glutathione peroxidases GPx1 and GPx4 being substantially reduced. Expression of the tumor suppressor genes BRCA1 and p53 was also altered in a contrasting manner in MMTV-Cre mice, suggesting greater susceptibility to cancer and/or increased cell apoptosis. Thus, the conditional Trsp knockout mouse allows tissue-specific manipulation of Sec tRNA and selenoprotein expression, suggesting that this approach will provide a useful tool for studying the role of selenoproteins in health

Interaction with PALB2 Is Essential for Maintenance of Genomic Integrity by BRCA2

<div><p>Human breast cancer susceptibility gene, <i>BRCA2</i>, encodes a 3418-amino acid protein that is essential for maintaining genomic integrity. Among the proteins that physically interact with BRCA2, Partner and Localizer of BRCA2 (PALB2), which binds to the N-terminal region of BRCA2, is vital for its function by facilitating its subnuclear localization. A functional redundancy has been reported between this N-terminal PALB2-binding domain and the C-terminal DNA-binding domain of BRCA2, which undermines the relevance of the interaction between these two proteins. Here, we describe a genetic approach to examine the functional significance of the interaction between BRCA2 and PALB2 by generating a knock-in mouse model of <i>Brca2</i> carrying a single amino acid change (Gly25Arg, <i>Brca2</i>^<i>G25R</i>) that disrupts this interaction. In addition, we have combined <i>Brca2</i>^<i>G25R</i> homozygosity as well as hemizygosity with <i>Palb2</i> and <i>Trp53</i> heterozygosity to generate an array of genotypically and phenotypically distinct mouse models. Our findings reveal defects in body size, fertility, meiotic progression, and genome stability, as well as increased tumor susceptibility in these mice. The severity of the phenotype increased with a decrease in the interaction between BRCA2 and PALB2, highlighting the significance of this interaction. In addition, our findings also demonstrate that hypomorphic mutations such as <i>Brca2</i>^<i>G25R</i> have the potential to be more detrimental than the functionally null alleles by increasing genomic instability to a level that induces tumorigenesis, rather than apoptosis.</p></div

Neuron-specific <i>Secp43</i> mutant mice.

<p>(A) <i>Secp43</i> mRNA levels measured by qPCR. Deletion of exons 7 and 8 was successful in neurons of <i>T</i>α<i>1-Cre; Secp43</i>^<i>fl/fl</i> mice (**p<0.01) Student’s <i>t</i> test. Number of animals: male controls (n = 5), male mutants (n = 4). (B) Selenoprotein expression in adult cerebral cortex is not altered as judged by Western blots against stress-related selenoproteins. N = 5 male animals for controls and <i>T</i>α<i>1-Cre; Secp43</i>^<i>fl/fl</i> mice. (C) Rotarod performance is reduced in adult <i>T</i>α<i>1-Cre; Secp43</i>^<i>fl/fl</i> mice. n = 10–16. Significant by genotype, two-way ANOVA. (D) Cerebellar histology is not altered. Normal foliation pattern and calbindin expression in Purkinje cells.</p

Increased tumor predisposition in <i>Brca2</i>^<i>G25R</i> hemizygous mice.

<p><b>A.</b> Median and average age of death, including number of mice per genotype analyzed (n) and shown by gender. <b>B.</b> Kaplan-Meier tumor-free survival curves of <i>Brca2</i> mutant animals (104 week study). <b>C.</b> Kaplan-Meier tumor-free survival curves of <i>Brca2</i> mutant mice on a <i>Trp53</i>^<i>Ko/+</i> genetic background (104 week study). <b>D.</b> and <b>E.</b> Distribution of tumor types found in mice of indicated genotypes. Abbreviations: NT = No tumor observed, BCL = B-cell lymphoma, TCL = T-cell lymphoma, HS = Histiocytic sarcoma, OS = Osteosarcoma, HemS = Hemangiosarcoma, OthSa = Other sarcomas, AD = Adenomas, OthCa = Other carcinomas, MT = Multiple tumors, UNK = cause of death unknown. * UNK are included in the tumor-free survival curves as potential neoplasias. Controls are represented by: Brca2^+/+ and Brca2^Ko/+. Abbreviations: Brca2^Ko/+ = <b>B2</b>^<b>Ko/+</b>, Brca2^G25R/+ = <b>B2</b>^<b>GR/+</b>, Brca2^G25R/G25R = <b>B2</b>^<b>GR/GR</b>, Brca2^G25R/Ko = <b>B2</b>^<b>GR/Ko</b>.</p

Phryma leptostachya L. var. oblongifolia Honda

原著和名: ナガバハエドクサウ科名: ハエドクソウ科 = Phrymaceae採集地: 愛知県 南設楽郡 鳳来町 槇原 (三河 南設楽郡 鳳来町 槇原)採集日: 1981/8/16採集者: 萩庭丈壽整理番号: JH023907国立科学博物館整理番号: TNS-VS-97390

Observed and expected birth ratio and Chi Square analysis of offspring of various genotypes from <i>Brca2</i>^<i>GR/GR</i> X <i>Brca2</i>^<i>Ko/+</i> cross.

<p>Observed and expected birth ratio and Chi Square analysis of offspring of various genotypes from <i>Brca2</i>^<i>GR/GR</i> X <i>Brca2</i>^<i>Ko/+</i> cross.</p