A Selectable and Excisable Marker System for the Rapid Creation of Recombinant Poxviruses

Genetic manipulation of poxvirus genomes through attenuation, or insertion of therapeutic genes has led to a number of vector candidates for the treatment of a variety of human diseases. The development of recombinant poxviruses often involves the genomic insertion of a selectable marker for purification and selection purposes. The use of marker genes however inevitably results in a vector that contains unwanted genetic information of no therapeutic value.Here we describe an improved strategy that allows for the creation of marker-free recombinant poxviruses of any species. The Selectable and Excisable Marker (SEM) system incorporates a unique fusion marker gene for the efficient selection of poxvirus recombinants and the Cre/loxP system to facilitate the subsequent removal of the marker. We have defined and characterized this new methodological tool by insertion of a foreign gene into vaccinia virus, with the subsequent removal of the selectable marker. We then analyzed the importance of loxP orientation during Cre recombination, and show that the SEM system can be used to introduce site-specific deletions or inversions into the viral genome. Finally, we demonstrate that the SEM strategy is amenable to other poxviruses, as demonstrated here with the creation of an ectromelia virus recombinant lacking the EVM002 gene.The system described here thus provides a faster, simpler and more efficient means to create clinic-ready recombinant poxviruses for therapeutic gene therapy applications

SIRT1 catalytic activity has little effect on tumor formation and metastases in a mouse model of breast cancer.

The protein deacetylase SIRT1 has been implicated in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. There are conflicting data that make it unclear whether Sirt1 functions as an oncogene or tumor suppressor. To assess the effect of SIRT1 on the emergence and progression of mammary tumors, we crossed mice that harbor a point mutation that abolishes SIRT1 catalytic activity with mice carrying the polyoma middle T transgene driven by the murine mammary tumor virus promoter (MMTV-PyMT). The absence of SIRT1 catalytic activity neither accelerated nor blocked the formation of tumors and metastases in this model. There was a lag in tumor latency that modestly extended survival in Sirt1 mutant mice that we attribute to a delay in mammary gland development and not to a direct effect of SIRT1 on carcinogenesis. These results are consistent with previous evidence suggesting that Sirt1 is not a tumor promoter or a tumor suppressor

Modulation of Tumorigenesis by Dietary Intervention Is Not Mediated by SIRT1 Catalytic Activity

<div><p>The protein deacetylase SIRT1 is involved in the regulation of a large number of cellular processes that are thought to be required for cancer initiation and progression. Both SIRT1 activity and tumorigenesis can be influenced by dietary fat and polyphenolics. We set out to determine whether dietary modulations of tumorigenesis are mediated by SIRT1 catalytic functions. We introduced a mammary gland tumor-inducing transgene, MMTV-PyMT, into stocks of mice bearing a H355Y point mutation in the <i>Sirt1</i> gene that abolishes SIRT1 catalytic activity. Tumor latency was reduced in animals fed a high fat diet but this effect was not dependent on SIRT1 activity. Resveratrol had little effect on tumor formation except in animals heterozygous for the mutant <i>Sirt1</i> gene. We conclude that the effects of these dietary interventions on tumorigenesis are not mediated by modulation of SIRT1 catalytic activity.</p></div

Abolition of SIRT1 enzymatic activity results in blunted ductal morphogenesis in the mammary gland.

<p>Right panel, representative photographs of whole mounts of the 4^th abdominal mammary gland in <i>Sirt1</i>^+/+, <i>Sirt1</i>^Y/+ and <i>Sirt1</i>^Y/Y mice at eleven weeks of age (scale bar, 5mm). Left panel, a higher magnification view of the ductal network in representative mammary gland whole mounts of the 4^th abdominal mammary gland in <i>Sirt1</i>^+/+, <i>Sirt1</i>^Y/+ and <i>Sirt1</i>^Y/Y mice at eleven weeks of age. 400X magnification (scale bar, 0.7 mm).</p

Expression of SIRT1, Middle T Antigen, and ERα protein in mammary tumors.

<p>Representative immunohistochemical staining for SIRT1 (A-C), Middle T Antigen (D-F), and ERα (G-I) in mammary tumors collected at humane endpoint from <i>Sirt1</i>^+/+, <i>Sirt1</i>^Y/+ and <i>Sirt1</i>^Y/Y mice at 200X magnification (scale bars, 100 μm).</p

Abrogation of SIRT1 catalytic activity does not prevent mammary tumor formation in the MMTV-PyMT mouse model of breast cancer.

<p><b>A</b>) Kaplan Meir plot showing the percentage of surviving animals over time. N= 10 mice per genotype. <i>Sirt1</i>^Y/Y animals had a significantly longer overall survival time than the <i>Sirt1</i>^+/+ and the <i>Sirt1</i>^Y/+ mice (p <0.01) <b>B</b>) Tumor burden as a proportion of total body weight at humane endpoint. All tumors were removed and weighed at necropsy. Points represent individual animals and bars represent the median. N=10 mice per genotype, all animals carried the MMTV-PyMT transgene.</p

High fat diet decreased tumor latency but did not affect survival in MMTV-PyMT transgenic mice.

<p><b>A, C, E)</b> Female mice of the indicated <i>Sirt1</i> genotypes carrying the MMTV-PyMT transgene were weaned onto high fat diet (HFD) or normal chow immediately following weaning. The mean number of mammary glands with a palpable mass was assessed at weekly intervals. Error bars indicate SEM. A significant difference in tumor latency was observed in <i>Sirt1</i>^+/+ (P<0.0001) and <i>Sirt1</i>^Y/Y mice (P<0.0001). <b>B, D, F</b>) Kaplan Meier plot of surviving animals. N = 10 mice per <i>Sirt1</i> genotype per diet (□ standard diet,▪ HFD).</p

Loss of SIRT1 catalytic activity does not affect expression of the PyMT transgene.

<p>Representative immunohistochemical staining for Middle T Antigen in mammary glands of PyMT⁺/<i>Sirt1</i>^+/+ and PyMT⁺/<i>Sirt1</i>^Y/Y mice collected at 6 weeks of age (scale bars equal to 100 μm). Arrows indicate areas of mammary intraepithelial neoplasia.</p

Loss of SIRT1 catalytic activity is associated with increase tumor latency

<p><b>A)</b> Kaplan Meir plot measuring the percentage of mice without any palpable mammary gland mass at the given age. N= 10 mice per genotype. There was a significant delay in the time at which the <i>Sirt1</i>^Y/Y developed their first detectable mass as compared to the <i>Sirt1</i>^+/+ and the <i>Sirt1</i>^Y/+ mice (P <0.01 and P < 0.05, respectively). <b>B)</b> The mean number of mammary glands with a palpable mass over time as measured at weekly intervals after birth. N= 10 mice per genotype. Error bars indicate SEM.</p