Lactation Defect in a Widely Used MMTV-Cre Transgenic Line of Mice

MMTV-Cre mouse lines have played important roles in our understanding about the functions of numerous genes in mouse mammary epithelial cells during mammary gland development and tumorigenesis. However, numerous studies have not included MMTV-Cre mice as controls, and many investigators have not indicated which of the different MMTV-Cre founder lines were used in their studies. Here, we describe a lactation defect that severely limits the use of one of the most commonly used MMTV-Cre founder lines.To explore the role of protein tyrosine phosphatase Shp1 in mammary gland development, mice bearing the floxed Shp1 gene were crossed with MMTV-Cre mice and mammary gland development was examined by histological and biochemical techniques, while lactation competency was assessed by monitoring pup growth. Surprisingly, both the Shp1fl/+;MMTV-Cre and MMTV-Cre female mice displayed a severe lactation defect when compared to the Shp1 fl/+ control mice. Histological and biochemical analyses reveal that female mice expressing the MMTV-Cre transgene, either alone or in combination with floxed genes, exhibit defects in lobuloalveolar expansion, presence of large cytoplasmic lipid droplets in luminal alveolar epithelial cells postpartum, and precocious induction of involution. Using a PCR-based genotyping method, the three different founder lines can be distinguished, and we determined that the MMTV-Cre line A, the most widely used MMTV-Cre founder line, exhibits a profound lactation defect that limits its use in studies on mammary gland development.The identification of a lactation defect in the MMTV-Cre line A mice indicates that investigators must use MMTV-Cre alone mice as control in studies that utilize Cre recombinase to excise genes of interest from mammary epithelial cells. Our results also suggest that previous results obtained in studies using the MMTV-Cre line A line should be re-evaluated if the controls did not include mice expressing only Cre recombinase

Influence of Doped H2O or H(2)on Soot Production and Power Capability in the Fuel-rich Gas Generator

Influence of doped H2O or H(2)on both soot production and power capability in the fuel-rich gas generator has been studied together by using the program of chemical equilibrium with applications (CEA). The oxidant is LOX, and the fuel is composed of Jet-A and the additive. The parameters of the gas generator are as follows: the range of combustion temperature is 800-1700 K, combustion pressure is 0.1-5.0 MPa, oxidant/fuel ratio is 0.1-1.2, and the mass percent of the additive in fuel is 0-60%. The results indicate that the addition of either H2O or H(2)can obviously reduce the mass percent of soot in combustion products, and the reasons are discussed on the base of the products distributions. Moreover, the minimum amounts of addition to surrender mass percent of soot less than 0.1% are present. The effects of combustion pressure on soot mass percent in combustion products appear turning appoints around 1100 K, no matter the additive is H2O or H-2. The addition of H(2)can obviously improve the power capacity of combustion products in the whole temperature range. The addition of H2O can also improve slightly the power capacity of combustion products, when the combustion temperature is less than 1400 K. Effective molar weight of combustion products is the main factor affecting power capacity

EFFECTS OF DESERT SAND SUBSTITUTION ON CONCRETE PROPERTIES USING DESERT SAND-DOPED QUARTZ SAND AS THE FINE AGGREGATE

As governments are increasingly attaching more importance to environmental protection, many policies and regulations are being promulgated to restrict or prohibit river sand extraction. Thus, there is a growing tendency for concrete production to use desert sand-doped quartz sand as the fine aggregate. This paper investigated the microstructure, mechanical properties, durability, and volume stability of concrete with desert sand and quartz sand. In the study, the strength grades of concrete ranged from C30 to C100, while the substitution ratios of the desert sand for the quartz sand ranged from 0% to 100%. The results showed that a high substitution ratio of desert sand increased the concrete porosity and extended the interface zone width. The concrete strength generally decreased as the desert sand substitution ratio increased, however, for the C50 and C60 strength grades, concrete with 20% desert sand had minor positive effects. Basically, as the desert sand substitution ratio increased, the Cl- penetration resistance and frost resistance tended to be weaker, although there were a few exceptions. High substitution ratios of desert sand were also not good for the sulfate attack resistance of concrete. In most cases, concrete with 20% desert sand shrank more slowly than concrete without desert sand. These conclusions provide the experimental support for the promotion of desert sand applications in the concrete industry

Hypergolic ignition modulated by head-on collision, intermixing and convective cooling of binary droplets with varying sizes

The hypergolic ignition induced by the head-on collision of TMEDA and WFNA droplets was experimentally investigated with emphasis on the effect of droplet size on the ignitibility and the ignition delay time. The ignitibility regime nomogram in We - d(o) space indicates that the reduction of droplet size tends to suppress the hypergolic ignition. The ignition delay time, which was precisely determined by using grayscale level analysis, becomes shorter for smaller droplets. The seemingly conflicting size effects were resolved by means of time scaling analysis to reveal the size dependence of the three pre-ignition processes, which were identified as the first stage of droplet collision, deformation and intermixing, the second stage of droplet heating from interior to surface, and the third stage of droplet vaporization subject to heat loss by convective cooling. (C) 2019 Elsevier Ltd. All rights reserved

Primers used for inverse PCR and genotyping the MMTV-Cre mice.

<p>Primers used for inverse PCR and genotyping the MMTV-Cre mice.</p

Changes in the activation of Stat5 and Stat3 in mammary glands from mice expressing the MMTV-Cre transgene during late pregnancy and early lactation.

<p>The number 4 mammary glands from Shp1 fl/+ (control) (lane 1), Shp1 fl/+;MMTV-Cre (lane 2), and MMTV-Cre (lane 3) mice at pregnancy day 15 (P15), lactation day 1 (L1), day 10 (L10), and from wild type FVB mice at involution day 2 (I2) (lane 4) were isolated and whole tissue lysates prepared as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#s4" target="_blank">Materials and Methods</a>. Equal amount of proteins (∼40 µg) from each sample were resolved by gel electrophoresis, and immunoblotted with antibodies against phospho-Stat5 (p-Stat5; Y694), or phospho-Stat3 (p-Stat3; Y705). The immunoblots were reprobed with antibodies against Stat5, Stat3, and cytokeratin-18 (CK-18), and beta-actin to demonstrate equal loading. Similar results were seen from at least two different mice for each genotype.</p

Impaired differentiation and precocious involution of mammary epithelial cells in mice expressing the MMTV-Cre transgene.

<p>(A) The number 4 mammary glands from Shp1 fl/+ (control), Shp1 fl/+/;MMTV-Cre, and MMTV-Cre mice were isolated from 10-week old virgin mice and subjected to whole mount analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#s4" target="_blank">Materials and Methods</a>. Scale bar = 5 mm. (B, C) Six-week old Shp1 fl/+ (control), Shp1 fl/+;MMTV-Cre, and MMTV-Cre female mice were mated with male wild type mice. The number 4 mammary glands were removed at pregnancy day 15 (P15), lactation day 1 (L1), and day 10 (L10), and processed for histological analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#s4" target="_blank">Materials and Methods</a>. Images were taken under lower (B) and higher (C) magnification respectively. Scale bars represent 100 µm in (B) and 50 µm in (C). Similar results were seen from at least two different mice for each genotype. (D, E) Analysis of apoptotic cells in L10 mammary glands as described in (B, C). (D) Sections of L10 mammary glands from indicated mice were stained with antibody against cleaved caspase 3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#s4" target="_blank">Materials and Methods</a>). Cells positive for cleaved caspase 3 are stained dark-brown. Images were taken using 40X objective lens. Scale bar = 50 µm. (E) Quantification of caspase 3 positive cells from (D) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#s4" target="_blank">Materials and Methods</a>). Two mice from each genotype were analyzed. **p<0.01 and ***p<0.001.</p

Identification of the linkage of the K14-Agouti cassette to the MMTV-Cre transgene using inverse PCR.

<p>(A) The schematic diagram of the inverse PCR. (B) PCR using specific primer sets identified the K14-agouti cassette adjacent to the MMTV-Cre transgene. The linkage of the linkage of the MMTV-Cre and K14-agouti transgenes allow distinction of the line A, line D and line F founder lines. The MMTV-Cre cassette <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#pone.0019233-Freeburn1" target="_blank">[5]</a> also contains a rabbit growth hormone intron sequence (RGI). The K14-Agouti cassette <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019233#pone.0019233-Kucera1" target="_blank">[14]</a> also contains a human growth hormone polyadenylation sequence (GHA). The Cre-F/Cre-R primer set identifies the presence of Cre gene. The K14P-3R/GHA-1F primer set identifies the presence of the K14-agouti cassette. The MMTV-1R/GHA-1R primer set identifies the presence of the head-to-tail linkage of the K14-agouti cassette and the MMTV-Cre cassette. The RGI-1F/GHA-1R primer set identifies the presence of the tail-to-tail linkage of the MMTV-Cre cassette and the K14-agouti cassette. 1, wild type DNA; 2, MMTV-Cre DNA used in this study; 3, MMTV-Cre line A DNA from Jackson lab; 4, MMTV-Cre line D from Jackson lab; 5, MMTV-Cre line A DNA from Hennighausen lab; 6, MMTV-Cre line F DNA from Hennighausen lab; N, no DNA control. Note, lane 2, 3, and 5 (all Line A) have the same PCR fragments for the head-tail and the tail-tail linkages whereas lane 4 (line D), and lane 6 (line F) only have PCR fragments for the tail-tail linkage. The sizes of PCR products are listed under each DNA gel panel.</p

Shp1 fl/+;MMTV-Cre and MMTV-Cre display a lactation defect.

<p>Shp1 fl/+ (control), Shp1 fl/+;MMTV-Cre, and MMTV-Cre female mice were mated with wild type FVB male mouse. On the first day following parturition, the litter size was normalized to 6 pups/litter. Pups were weighted at lactation days 5, 8, and 10. The average weight of each litter is shown and bars indicate the standard deviation (SD). ** indicates P<0.001. The data shown is representative of at least two independent experiments.</p

Protein-tyrosine Phosphatase PTPN9 Negatively Regulates ErbB2 and Epidermal Growth Factor Receptor Signaling in Breast Cancer Cells*

ErbB family of the receptor protein-tyrosine kinase plays an important role in the progression of human cancers including breast cancer. Finding protein-tyrosine phosphatase (PTPs) that can specifically regulate the function of ErbB should help design novel therapies for treatment. By performing a small interfering RNA screen against 43 human PTPs, we find that knockdown of protein-tyrosine phosphatase PTPN9 significantly increases ErbB2 tyrosyl phosphorylation in the SKBR3 breast cancer cell line. In addition, knockdown of PTPN9 expression also enhances tyrosyl phosphorylation of the ErbB1/epidermal growth factor receptor (EGFR) in the MDA-MB-231 breast cancer cell line. Conversely, increasing expression of PTPN9 wild type (WT) inhibits tyrosyl phosphorylation of ErbB2 and EGFR. To test whether ErbB2 and EGFR are substrates of PTPN9, PTPN9 WT, and a substrate trapping mutant (PTPN9 DA) are overexpressed in SKBR3 and MDA-MB-231 cells. Compared with vector control, expression of PTPN9 WT significantly inhibits whereas expression of PTPN9 DA dramatically enhances tyrosyl phosphorylation of ErbB2 and EGFR, respectively. In contrast, expression of PTPN9 WT or DA mutant does not affect tyrosyl phosphorylation of ErbB3 and Shc. Importantly, coimmunoprecipitation and glutathione S-transferase fusion protein pulldown experiments show that tyrosol-phosphorylated ErbB2 or EGFR is preferentially associated with PTPN9 DA compared with PTPN9 WT, indicating that ErbB2 and EGFR are substrates of PTPN9. Furthermore, PTPN9 WT expression specifically impairs EGF-induced STAT3 and STAT5 activation, and inhibits the cell growth in soft agar. Last, PTPN9 WT expression also reduces invasion and MMP2 expression of MDA-MB-231 cells. Our data suggest PTPN9 as a negative regulator of breast cancer cells by targeting ErbB2 and EGFR and inhibiting STAT activation