Cul4A is essential for spermatogenesis and male fertility

The mammalian Cul4 genes, Cul4A and Cul4B, encode the scaffold components of the cullin-based E3 ubiquitin ligases. The two Cul4 genes are functionally redundant. Recent study indicated that mice expressing a truncated CUL4A that fails to interact with its functional partner ROC1 exhibit no developmental phenotype. We generated a Cul4A−/− strain lacking exons 4–8 that does not express any detectable truncated protein. In this strain, the male mice are infertile and exhibit severe deficiencies in spermatogenesis. The primary spermatocytes are deficient in progression through late prophase I, a time point when expression of the X-linked Cul4B gene is silenced due to meiotic sex chromosome inactivation. Testes of the Cul4A−/− mice exhibit extensive apoptosis. Interestingly, the pachytene spermatocytes exhibit persistent double stranded breaks, suggesting a deficiency in homologous recombination. Also, we find that CUL4A localizes to the double stranded breaks generated in pre-pachytene spermatocytes. The observations identify a novel function of CUL4A in meiotic recombination and demonstrate an essential role of CUL4A in spermatogenesis.     

Cul4A overexpression associated with Gli1 expression in malignant pleural mesothelioma

Malignant pleural mesothelioma (mesothelioma) is a highly aggressive cancer without an effective treatment. Cul4A, a scaffold protein that recruits substrates for degradation, is amplified in several human cancers, including mesothelioma. We have recently shown that Cul4A plays an oncogenic role in vitro and in a mouse model. In this study, we analysed clinical mesothelioma tumours and found moderate to strong expression of Cul4A in 70.9% (51/72) of these tumours, as shown by immunohistochemistry. In 72.2% mesothelioma tumours with increased Cul4A copy number identified by fluorescence in situ hybridization analysis, Cul4A protein expression was moderate to strong. Similarly, Cul4A was overexpressed and Cul4A copy number was increased in human mesothelioma cell lines. Because Gli1 is highly expressed in human mesothelioma cells, we compared Cul4A and Gli1 expression in mesothelioma tumours and found their expression associated (P < 0.05, chi‐square). In mesothelioma cell lines, inhibiting Cul4A by siRNA decreased Gli1 expression, suggesting that Gli1 expression is, at least in part, regulated by Cul4A in mesothelioma cells. Our results suggest a linkage between Cul4A and Gli1 expression in human mesothelioma.     

Cul4A is an oncogene in malignant pleural mesothelioma

Cullin 4A (Cul4A) is important in cell survival, development, growth and the cell cycle, but its role in mesothelioma has not been studied. For the first time, we identified amplification of the Cul4A gene in four of five mesothelioma cell lines. Consistent with increased Cul4A gene copy number, we found that Cul4A protein was overexpressed in mesothelioma cells as well. Cul4A protein was also overexpressed in 64% of primary malignant pleural mesothelioma (MPM) tumours. Furthermore, knockdown of Cul4A with shRNA in mesothelioma cells resulted in up‐regulation of p21 and p27 tumour suppressor proteins in a p53‐independent manner in H290, H28 and MS‐1 mesothelioma cell lines. Knockdown of Cul4A also resulted in G0/G1 cell cycle arrest and decreased colony formation in H290, H28 and MS‐1 mesothelioma cell lines. Moreover, G0/G1 cell cycle arrest was partially reversed by siRNA down‐regulation of p21 and/or p27 in Cul4A knockdown H290 cell line. In the contrary, overexpression of Cul4A resulted in down‐regulation of p21 and p27 proteins and increased colony formation in H28 mesothelioma cell line. Both p21 and p27 showed faster degradation rates in Cul4A overexpressed H28 cell line and slower degradation rates in Cul4A knockdown H28 cell line. Our study indicates that Cul4A amplification and overexpression play an oncogenic role in the pathogenesis of mesothelioma. Thus, Cul4A may be a potential therapeutic target for MPM.     

Generation of mice with conditional null allele for GdX/Ubl4A

GdX (also named Ubl4A) is a house-keeping gene located on the X chromosome and encodes a protein harboring an ubiquitin-like domain in human and mouse. Although identified in 1988, the function of GdX remains unknown. To elucidate the role of GdX in vivo, we generated a conditional GdX knockout mouse in which Exon 2 was flanked by two loxP sites. We obtained viable and fertile mice with homozygous GdX(flox/flox) or GdX(flox/Y) allele. Germ-line transmission was confirmed by crossing the mouse bearing conditionally targeted allele with an EIIα-Cre transgenic mouse. GdX was successfully depleted in tissues of EIIα-Cre-GdX-null mice. GdX(-/-) and GdX(-/Y) mice are viable and exhibit normal development compared with wild-type littermates within 6 months during our observation. We also observed that GdX knockout male mice were functionally normal in the reproductive system where Ubl4B was specifically expressed. GdX(flox/flox) and GdX(flox/Y) conditional mice provide a tool for further tissue-specific function analysis of the GdX protein under different conditions.     

Transgenic Cre expression mice for generation of erythroid-specific gene alterations

Transgenic mice that express Cre recombinase in erythroid cell lineages were developed so that genes affecting erythropoiesis/hematopoiesis may be altered without necessarily affecting fetus viability. A micro-LCR cassette-beta-globin promoter-Cre recombinase gene (microLCR-betapr-Cre) construct was synthesized and used to generate transgenic mice. Concurrently, we produced mice containing a microLCR-loxP-flanked beta sickle gene (microLCR-loxP-beta(S)-loxP) construct. microLCR-betapr-Cre mice with intact transgenes in variable copy number were identified. Cre expression was assessed by RNAse protection and RT-PCR. Cre function was ascertained by breeding to microLCR-loxP-beta(S)-loxP mice. We demonstrate that beta(S) expression was not detected in the blood of bigenics, but the gene was present in nonerythroid cells. Thus, excision of the loxP-flanked beta(S) gene was restricted to erythroid cell lineages.     

Generation of a reporter mouse line expressing Akt and EGFP upon Cre-mediated recombination

The serine-threonine kinase Akt regulates multiple biological processes. An important strategy to study Akt signaling in different tissues is targeted activation of this pathway in vivo. The current studies describe the generation of a mouse model that combines a double reporter system with activation of a constitutively active form of Akt1 (caAkt) in a Cre-dependent manner. Before Cre recombination, these mice express LacZ during development as well as in most adult tissues. After Cre-mediated excision of the LacZ reporter, functionality of the transgene was demonstrated by expression of the caAkt mutant along with the second reporter, EGFP in different pancreatic compartments and in the nervous system. This animal model provides a critical reagent for assessing the effects of Akt activation in specific tissues. The lineage-tracing properties provide a useful tool to study the role of Akt signaling in regulation of differentiation programs during development and plasticity of mature tissues.     

Atp4b promoter directs the expression of Cre recombinase in gastric parietal cells of transgenic mice

Parietal cells are one of the largest epithelium cells of the mucous membrane of the stomach that secrete hydrochloric acid.To study the function of gastric parietal cells during gastric epithelium homeostasis,we generated a transgenie mouse line,namely,Atp4b-Cre,in which the expression of Cre recombinase was controlled by a 1.0 kb promoter of mouse β-subunit of H+-,K+-ATPase gene(Atp4b).In order to test the tissue distribution and excision activity of Cre recombinase in vivo,the Atp4b-Cre transgenic mice were bred with the reporter strain ROSA26 and a mouse strain that carries Smad4 conditional alleles(Smad4Co/Co).Multiple-tissue PCR of Atp4b-Cre;Smad4Co/+mice revealed that the recombination only happened in the stomach.As indicated by LacZ staining,ROSA26;Atp4b-Cre double transgenic mice showed efficient expression of Cre recombinase within the gastric parietal cells.These results showed that this Atp4b-Cre mouse line could be used as a powerful tool to achieve conditional gene knockout in gastric parietal cells.     

Knockdown of cullin 4A inhibits growth and increases chemosensitivity in lung cancer cells

Cullin 4A (Cul4A) has been observed to be overexpressed in various cancers. In this study, the role of Cul4A in the growth and chemosensitivity in lung cancer cells were studied. We showed that Cul4A is overexpressed in lung cancer cells and tissues. Knockdown of the Cul4A expression by shRNA in lung cancer cells resulted in decreased cellular proliferation and growth in lung cancer cells. Increased sensitivity to gemcitabine, a chemotherapy drug, was also noted in those Cul4A knockdown lung cancer cells. Moreover, increased expression of p21, transforming growth factor (TGF)‐β inducible early gene‐1 (TIEG1) and TGF beta‐induced (TGFBI) was observed in lung cancer cells after Cul4A knockdown, which may be partially related to increased chemosensitivity to gemcitabine. G0/G1 cell cycle arrest was also noted after Cul4A knockdown. Notably, decreased tumour growth and increased chemosensitivity to gemcitabine were also noted after Cul4A knockdown in lung cancer xenograft nude mice models. In summary, our study showed that targeting Cul4A with RNAi or other techniques may provide a possible insight to the development of lung cancer therapy in the future.     

Inducible gene deletion in the entire cardiac conduction system using Hcn4‐CreERT2 BAC transgenic mice

Developmental defects and disruption of molecular pathways of the cardiac conduction system (CCS) can cause life‐threatening cardiac arrhythmias. Despite decades of effort, knowledge about the development and molecular control of the CCS remains primitive. Mouse genetics, complementary to other approaches such as human genetics, has become a key tool for exploring the developmental processes of various organs and associated diseases. Genetic analysis using mouse models will likely provide great insights about the development of the CCS, which can facilitate the development of novel therapeutic strategies to treat arrhythmias. To enable genetic studies of the CCS, CCS‐associated Cre mouse models are essential. However, existing mouse models with Cre activity reported in the CCS have various limitations such as Cre leak, haploinsufficiency, and inadequate specificity of the Cre activity. To circumvent those limitations, we successfully generated Hcn4‐CreERT2 bacterial artificial chromosome (BAC) transgenic mice using BAC recombineering in which Cre activity was specifically detected in the entire CCS after tamoxifen induction. Our Hcn4‐CreERT2 BAC transgenic line will be an invaluable genetic tool with which to dissect the developmental control of CCS and arrhythmias. genesis 52:134–140. © 2013 Wiley Periodicals, Inc.     

Textpresso site‐specific recombinases: A text‐mining server for the recombinase literature including Cre mice and conditional alleles

Textpresso Site Specific Recombinases ( http://ssrc.genetics.uga.edu/ ) is a text‐mining web server for searching a database of more than 9,000 full‐text publications. The papers and abstracts in this database represent a wide range of topics related to site‐specific recombinase (SSR) research tools. Included in the database are most of the papers that report the characterization or use of mouse strains that express Cre recombinase as well as papers that describe or analyze mouse lines that carry conditional (floxed) alleles or SSR‐activated transgenes/knockins. The database also includes reports describing SSR‐based cloning methods such as the Gateway or the Creator systems, papers reporting the development or use of SSR‐based tools in systems such as Drosophila, bacteria, parasites, stem cells, yeast, plants, zebrafish, and Xenopus as well as publications that describe the biochemistry, genetics, or molecular structure of the SSRs themselves. Textpresso Site Specific Recombinases is the only comprehensive text‐mining resource available for the literature describing the biology and technical applications of SSRs. genesis 47:842–846, 2009. © 2009 Wiley‐Liss, Inc.     

Generation of Elf5‐Cre knockin mouse strain for trophoblast‐specific gene manipulation

Placental development is a complex and highly controlled process during which trophoblast stem cells differentiate to various trophoblast subtypes. The early embryonic death of systemic gene knockout models hampers the investigation of these genes that might play important roles during placentation. A trophoblast specific Cre mouse model would be of great help for dissecting out the potential roles of these genes during placental development. For this purpose, we generate a transgenic mouse with the Cre recombinase inserted into the endogenous locus of Elf5 gene that is expressed specifically in placental trophoblast cells. To analyze the specificity and efficiency of Cre recombinase activity in Elf5‐Cre mice, we mated Elf5‐Cre mice with Rosa26^mT/mG reporter mice, and found that Elf5‐Cre transgene is expressed specifically in the trophoectoderm as early as embryonic day 4.5 (E4.5). By E12.5, the activity of Elf5‐Cre transgene was detected exclusively in all derivatives of trophoblast lineages, including spongiotrophoblast, giant cells, and labyrinth trophoblasts. In addition, Elf5‐Cre transgene was also active during spermatogenesis, from spermatids to mature sperms, which is consistent with the endogenous Elf5 expression in testis. Collectively, our results provide a unique tool to delete specific genes selectively and efficiently in trophoblast lineage during placentation.     

Development of a general‐purpose method for cell purification using Cre/loxP‐mediated recombination

A mammalian body is composed of more than 200 different types of cells. The purification of a certain cell type from tissues/organs enables a wide variety of studies. One popular cell purification method is immunological isolation, using antibodies against specific cell surface antigens. However, this is not a general‐purpose method, since suitable antigens have not been found in certain cell types, including embryonic gonadal somatic cells and Sertoli cells. To address this issue, we established a knock‐in mouse line, named R26 KI, designed to express the human cell surface antigen hCD271 through Cre/loxP‐mediated recombination. First, we used the R26 Kl mouse line to purify embryonic gonadal somatic cells. Gonadal somatic cells were purified from the R26 KI; Nr5a1‐Cre‐transgenic (tg) embryos almost equally as efficiently as from Nr5a1‐hCD271‐tg embryos. Second, we used the R26 KI mouse line to purify Sertoli cells successfully from R26 KI; Amh‐Cre‐tg testes. In summary, we propose that the R26 KI mouse line is a powerful tool for the purification of various cell types. genesis 53:387–393, 2015. © 2015 Wiley Periodicals, Inc.     

Elevated S100A4 in asthmatics and an allergen-induced mouse asthma model

The elevated S100A4 level has been found in some inflammatory diseases. However, the expression and role of S100A4 in asthma is unknown. The expression of S100A4 in induced sputum and plasma from healthy control and asthmatics were assessed by ELISA. Then an allergen-induced asthma mouse model treatment with anti-S100A4 antibody was used to explore the role of S100A4 in the pathogenesis of asthma. The S100A4 levels in sputum not in plasma in asthmatics were significantly increased than those of healthy controls and were negatively correlated with some lung function parameters and were positively correlated with sputum eosinophilia and lymphocyte. The expression of S100A4 in the lung as well as in BALF were also significantly higher in the asthma mouse model and treatment with anti-S100A4 antibody exhibited reductions in inflammatory cell accumulation, inflammatory mediators, and airway hyper-responsiveness. We further showed that LY294002, a specific inhibitor of PI3K, markedly decreased S100A4 expression in lung and S100A4 secretion in BALF in asthmatic mice. In conclusion, these data demonstrated that S100A4 may be involved in the pathogenesis of airway inflammation in asthma.     

Thyrocyte-specific expression of Cre recombinase in transgenic mice

A transgenic mouse line that expresses Cre recombinase under control of the human thyroid peroxidase (TPO) gene promoter was established. The activity and specificity of the TPO-driven Cre recombinase were examined by using Northern blotting and by crossing with the ROSA26 reporter transgenic mouse line. In the latter mice, Cre-mediated recombination occurred only in the thyrocytes, and recombination commenced around embryonic day 14.5, at the time during thyroid organogenesis when TPO expression begins. This study demonstrates that the TPO-Cre transgenic mouse is a powerful tool to specifically delete loxP-inserted (floxed) genes in thyrocytes and will be of great value in the study of thyrocyte-specific genes during development and/or in adult thyroids.     

特异性检测胰腺组织表达Cre重组酶转基因小鼠的方法

利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.