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The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

ObjectivesTo develop a mouse model for multispectral fluorescence imaging of the pancreas and pancreatic microenvironment.MethodsCre/loxP technology was used to develop this model. We crossed mT/mG indicator mice, engineered to constitutively express a conditional tdTomato transgene that converts to green fluorescent protein (GFP) expression after exposure to Cre recombinase, with Pdx1-Cre transgenic mice. To characterize this model for studies of pancreas biology, we performed bright light and fluorescence imaging of body cavities and intact organs and confocal microscopy of pancreata from offspring of Pdx1-Cre and mT/mG crosses.ResultsPdx1-Cre-mT/mG mice demonstrated bright GFP expression within the pancreas and duodenum and intense tdTomato expression in all other organs. Green fluorescent protein expression was mosaic in Pdx1-Cre-mT/mG pancreata, with most showing extensive conversion from tdTomato to GFP expression within the epithelial-derived elements of the pancreatic parenchyma. Because both GFP and tdTomato are membrane targeted, individual cell borders were clearly outlined in confocal images of mT/mG pancreata.ConclusionsThis mouse model enables multispectral fluorescence imaging of individual cells and cell processes at the microscopic level of the pancreatic microenvironment; it should prove valuable for a variety of fluorescence imaging studies, ranging from pancreatic development to pancreatic cancer biology.

Plectin, a high-molecular-weight cytoskeletal linker protein, binds with high affinity to intermediate filaments of all types and connects them to junctional complexes, organelles, and inner membrane systems. In addition, it interacts with actomyosin structures and microtubules. As a multifunctional protein, plectin has been implicated in several multisystemic diseases, the most common of which is epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). A great part of our knowledge about plectin’s functional diversity has been gained through the analysis of a unique collection of transgenic mice that includes a full (null) knockout (KO), several tissue-restricted and isoform-specific KOs, three double KOs, and two knock-in lines. The key molecular features and pathological phenotypes of these mice will be discussed in this review. In summary, the analysis of the different genetic models indicated that a functional plectin is required for the proper function of striated and simple epithelia, cardiac and skeletal muscle, the neuromuscular junction, and the vascular endothelium, recapitulating the symptoms of humans carrying plectin mutations. The plectin-null line showed severe skin and muscle phenotypes reflecting the importance of plectin for hemidesmosome and sarcomere integrity; whereas the ablation of individual isoforms caused a specific phenotype in myofibers, basal keratinocytes, or neurons. Tissue-restricted ablation of plectin rendered the targeted cells less resilient to mechanical stress. Studies based on animal models other than the mouse, such as zebrafish and C. elegans, will be discussed as well.

Development of external genitalia and perineum is the subject of developmental biology as well as toxicology and teratology researches. Cloaca forms in the lower (caudal) end of endoderm. Such endodermal epithelia and surrounding mesenchyme interact with various signals to form the external genitalia. External genitalia (the anlage termed as genital tubercle: GT) formation shows prominent sexually dimorphic morphogenesis in late embryonic stages, which is an unexplored developmental research field because of many reasons. External genitalia develop adjacent to the cloaca which develops urethra and corporal bodies. Developmental regulators including growth factor signals are necessary for epithelia‐mesenchyme interaction (EMI) in posterior embryos including the cloaca and urethra in the genitalia. In the case of male type urethra, formation of tubular urethra proceeds from the lower (ventral) side of external genitalia as a masculinization process in contrast to the case of female urethra. Mechanisms for its development are not elucidated yet due to the lack of suitable mutant mouse models. Because of the recent progresses of Cre (recombinase)‐mediated conditional target gene modification analyses, many developmental regulatory genes become increasingly analyzed. Conditional gene knockout mouse approaches and tissue lineage approaches are expected to offer vital information for such sexually dimorphic developmental processes. This review aims to offer recent updates on the progresses of these emerging developmental processes for the research field of congenital anomalies. [ABSTRACT FROM AUTHOR]

Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Supported primarily by the decrease of substrate receptor (SR) proteins of CRLs in cells deficient of a CSN subunit, CSN-mediated cullin deneddylation is believed to prevent autoubiquitination and self-destruction of the SR in active CRLs. However, it is unclear whether the decrease in SRs is solely due to protein destabilization. Moreover, our prior studies have demonstrated that cardiac specific knockout of Cops8 (Cops8-CKO) impairs autophagosome maturation and causes massive necrosis in cardiomyocytes but the underlying mechanism remains poorly understood. Given that Cops8 is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal Cops8-CKO and to explore their functional implications.Methods and Results: Myocardial transcriptomes of Cops8flox/flox, Cops8flox/+::Myh6-Cre, and Cops8flox/flox::Myh6-Cre littermate mice at postnatal 2 and 3 weeks were analyzed. The data were imported into an in-house analysis pipeline using Bioconductor for quantile normalization and statistical analysis. Differentially expressed genes (DEGs) between groups at each time point or between time points within the group were revealed by t-test. Genes with p < 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant DEGs. We found that (1) the Ingenuity Pathway Analysis (IPA) revealed significant enrichment of DEGs in multiple pathways, especially those responding to oxidative stress, in homozygous Cops8-CKO hearts at both 2 and 3 weeks, corroborating the occurrence of massive cardiomyocyte necrosis at 3 weeks; (2) the decreases in multiple CRL SR proteins were associated with decreased transcript levels; and (3) enrichment of DEGs in the chromatin remodeling pathway and the microtubule motility and vesicle trafficking pathways.Conclusions: Our data are consistent with the notion that Cops8/CSN plays a role in the transcriptional regulation of CRL SRs and in the redox and vesicle trafficking pathways.

Background/Aims: Optimal use of Cre mediated recombination in conditional animal models depends on well characterized Cre driver lines. Unfortunately, some Cre driver lines exhibit unexpected expression patterns hindering their utility in Cre/loxP systems. Thus, systematic assessment of new Cre lines is essential for generating useful Cre driver lines for future studies. Methods: Here, we describe a Cre Transgenic (Tg) mini-pig line in which the expression of Cre is directed by a 3-kb 5' fragment of the kidney-specific aquaporin 2 (AQP2); however, the AQP2-Cre Tg mini-pig line exhibits expression of Cre in alveolar epithelial cells (AECs) instead of collecting duct cells. The specificity of the AQP2-Cre plasmid was validated in vitro, and indicating that the AQP2-Cre was specifically expressed in the transfected LLC-PK1 cells. Absolute quantitative real-time PCR (qRT-PCR) and inverse PCR were performed to determine the copy numbers and integration sites of the AQP2-Cre transgene. Relative qRT-PCR was performed to evaluate variation in Cre expression levels over time. Results: Our data indicated that this AQP2-Cre Tg mini-pig line exhibits stable expression of Cre recombinase over time and in subsequent generations, even though the AQP2-Cre transgene was segregated and reduced in subsequent generations. Conclusion: Combined with our previous studies of the activity of this Cre, we conclude that this Cre Tg mini-pig line will provide a reliable tool for generating lung-specific gene targeting mini-pig models, thereby allowing the investigation of gene functions in lung development and studying the molecular mechanisms of human lung disease.

Insulin-like growth factor 1 ( Igf1) is important for skin development and homoeostasis. However, overexpression and inactivation studies have produced variable findings regarding its role in hair follicle (HF) biology. Here, we studied a conditional and inducible knockout of the Igf1 receptor ( Igf1r) in keratin 15-expressing bulge cells. Deletion of Igf1r after the development of the skin appendages in K15- Igf1rKO mice showed no abnormalities in epidermal homoeostasis. Numbers of bulge cells were lower in K15- Igf1rKO mice than in controls, without consequences on wound healing, at least in young mice. K15- Igf1rKO HFs entered anagen phase earlier than controls and showed a delay in the anagen/catagen switch. The expression of Bmp-4 mRNA was inhibited in HFs from K15- Igf1rKO. MED1 transcription was impaired in the epidermis of K15- Igf1rKO mice. These findings suggest that Igf1r controls the hair cycle, partly through Bmp-4 activation. [ABSTRACT FROM AUTHOR]

Background: The COP9 signalosome (CSN) consisting of 8 unique protein subunits (COPS1 through COPS8) serves as the cullin deneddylase, regulating the catalytic dynamics of cullin RING ligases (CRLs), the largest family of ubiquitin ligases. Supported primarily by the decrease of substrate receptor (SR) proteins of CRLs in cells deficient of a CSN subunit, CSN-mediated cullin deneddylation is believed to prevent autoubiquitination and self-destruction of the SR in active CRLs. However, it is unclear whether the decrease in SRs is solely due to protein destabilization. Moreover, our prior studies have demonstrated that cardiac specific knockout of Cops8 (Cops8-CKO) impairs autophagosome maturation and causes massive necrosis in cardiomyocytes but the underlying mechanism remains poorly understood. Given that Cops8 is nucleus-enriched and a prior report showed its binding to the promoter of several genes and association of its ablation with decreased mRNA levels of these genes, we sought to determine the dynamic changes of myocardial transcriptome in mice with perinatal Cops8-CKO and to explore their functional implications. Methods and Results: Myocardial transcriptomes of Cops8flox/flox, Cops8flox/+::Myh6-Cre, and Cops8flox/flox::Myh6-Cre littermate mice at postnatal 2 and 3 weeks were analyzed. The data were imported into an in-house analysis pipeline using Bioconductor for quantile normalization and statistical analysis. Differentially expressed genes (DEGs) between groups at each time point or between time points within the group were revealed by t-test. Genes with p < 0.05 after Benjamini and Hochberg false discovery rate correction for multiple hypothesis testing were considered as significant DEGs. We found that (1) the Ingenuity Pathway Analysis (IPA) revealed significant enrichment of DEGs in multiple pathways, especially those responding to oxidative stress, in homozygous Cops8-CKO hearts at both 2 and 3 weeks, corroborating the occurrence of massive cardiomyocyte necrosis at 3 weeks; (2) the decreases in multiple CRL SR proteins were associated with decreased transcript levels; and (3) enrichment of DEGs in the chromatin remodeling pathway and the microtubule motility and vesicle trafficking pathways. Conclusions: Our data are consistent with the notion that Cops8/CSN plays a role in the transcriptional regulation of CRL SRs and in the redox and vesicle trafficking pathways. [ABSTRACT FROM AUTHOR]

Medullary thymic epithelial cells (mTECs) contribute to self-tolerance by expressing and presenting peripheral tissue antigens for negative selection of autoreactive T cells and differentiation of natural regulatory T cells. The molecular control of mTEC development remains incompletely understood. We here demonstrate by TEC-specific gene manipulation in mice that the NF-κB transcription factor subunit RelB, which is activated by the alternative NF-κB pathway, regulates development of mature mTECs in a dose-dependent manner. Mice with conditional deletion of Relb lacked mature mTECs and developed spontaneous autoimmunity. In addition, the NF-κB subunits RelA and c-Rel, which are both activated by classical NF-κB signaling, were jointly required for mTEC differentiation by directly regulating the transcription of Relb . Our data reveal a crosstalk mechanism between classical and alternative NF-κB pathways that tightly controls the development of mature mTECs to ensure self-tolerance. [ABSTRACT FROM AUTHOR]

In humans, alterations in thyroid hormone signalling are associated with mood and anxiety disorders, but the neural mechanisms underlying such association are poorly understood. The present study investigates the involvement of neuronal thyroid hormone receptor α (TRα) in anxiety, using mouse genetics and Cre/loxP technology to specifically alter TRα signalling in neurons. We evaluated the behaviour of mice expressing a dominant negative, neuron-specific mutation of TRα ( TRα AMI /Cre3 mice), using the elevated-plus maze, light-dark box and open-field tests. In a first experiment, mice were housed individually, and the behaviour of TRα AMI /Cre3 mice differed significantly from that of control littermates in these 3 tests, suggesting heightened anxiety. In a second experiment, designed to evaluate the robustness of the results with the same 3 tests, mice were housed in groups. In these conditions, the behaviour of TRα AMI /Cre3 mice differed from that of control littermates only in the light-dark box. Thus, TRα AMI /Cre3 mice appear to be more likely to develop anxiety under stressful housing conditions than control mice. These results suggest that in adult mice, thyroid hormone signalling in neurons, via TRα, is involved in the control of anxiety behaviour. [ABSTRACT FROM AUTHOR]

The properties of CD4 + regulatory T cell (Treg) subsets are dictated by distinct patterns of gene expression determined by FOXP3 and different combinations of various transcription factors. Here we show the NF-κB transcription factor RelA is constitutively active in naïve and effector Tregs. The conditional inactivation of Rela in murine FOXP3 + cells induces a rapid onset, multi-focal autoimmune disease that depends on RelA being expressed in conventional T cells. In addition to promoting Treg lineage stability, RelA determines the size of the effector Treg population, a function influenced by the presence or absence of RelA in conventional T cells. These findings showing that RelA controls Treg stability and promotes the competitive fitness of effector Tregs highlight the importance of RelA activity in peripheral Treg induced tolerance. [ABSTRACT FROM AUTHOR]

Spermatogonial stem cells (SSCs) are a subpopulation of undifferentiated spermatogonia located in a niche at the base of the seminiferous epithelium delimited by Sertoli cells and peritubular myoid (PM) cells. SSCs self-renew or differentiate into spermatogonia that proliferate to give rise to spermatocytes and maintain spermatogenesis. Glial cell line-derived neurotrophic factor (GDNF) is essential for this process. Sertoli cells produce GDNF and other growth factors and are commonly thought to be responsible for regulating SSC development, but limited attention has been paid to the role of PM cells in this process. A conditional knockout (cKO) of the androgen receptor gene in PM cells resulted in male infertility. We found that testosterone (T) induces GDNF expression in mouse PM cells in vitro and neonatal spermatogonia (including SSCs) co-cultured with T-treated PM cells were able to colonize testes of germ cell-depleted mice after transplantation. This strongly suggested that T-regulated production of GDNF by PM cells is required for spermatogonial development, but PM cells might produce other factors in vitro that are responsible. In this study, we tested the hypothesis that production of GDNF by PM cells is essential for spermatogonial development by generating mice with a cKO of the Gdnf gene in PM cells. The cKO males sired up to two litters but became infertile due to collapse of spermatogenesis and loss of undifferentiated spermatogonia. These studies show for the first time, to our knowledge, that the production of GDNF by PM cells is essential for undifferentiated spermatogonial cell development in vivo. [ABSTRACT FROM AUTHOR]

Background/Aims: Optimal use of Cre mediated recombination in conditional animal models depends on well characterized Cre driver lines. Unfortunately, some Cre driver lines exhibit unexpected expression patterns hindering their utility in Cre/loxP systems. Thus, systematic assessment of new Cre lines is essential for generating useful Cre driver lines for future studies. Methods: Here, we describe a Cre Transgenic (Tg) mini-pig line in which the expression of Cre is directed by a 3-kb 5' fragment of the kidney-specific aquaporin 2 (AQP2); however, the AQP2-Cre Tg mini-pig line exhibits expression of Cre in alveolar epithelial cells (AECs) instead of collecting duct cells. The specificity of the AQP2-Cre plasmid was validated in vitro, and indicating that the AQP2-Cre was specifically expressed in the transfected LLC-PK1 cells. Absolute quantitative real-time PCR (qRT-PCR) and inverse PCR were performed to determine the copy numbers and integration sites of the AQP2-Cre transgene. Relative qRT-PCR was performed to evaluate variation in Cre expression levels over time. Results: Our data indicated that this AQP2-Cre Tg mini-pig line exhibits stable expression of Cre recombinase over time and in subsequent generations, even though the AQP2-Cre transgene was segregated and reduced in subsequent generations. Conclusion: Combined with our previous studies of the activity of this Cre, we conclude that this Cre Tg mini-pig line will provide a reliable tool for generating lung-specific gene targeting mini-pig models, thereby allowing the investigation of gene functions in lung development and studying the molecular mechanisms of human lung disease. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

This review describes the building and scientific activity of the Im-munology Department at the Institute for Genetics in Cologne, co-founded by Max Delbrück in post-World War II Germany. The pro-tagonist, a child of Russian emigrants, became interested in antibodies as a postdoc at the Pasteur Institute in Paris and a proponent of the antigen-bridge model of T-B cell collaboration during his early time in Cologne. He was challenged by the gap between cellular immunol-ogy and molecular genetics and profited from the advances of the latter as well as postwar economic growth in Germany. The Immunology Department became a place, and little universe in itself, where young scientists from all over the world came together to study cellular and molecular mechanisms of antibody formation. This included work on normal and malignant B cells in the human, particularly the origin of Hodgkin lymphoma, but the main focus was on B cell development and homeostasis, the germinal center reaction, and immunological mem-ory, developing recombinase-assisted and conditional gene targeting in mice as a main technical tool. [ABSTRACT FROM AUTHOR]

Background Angiotensin I-converting enzyme inhibitors and angiotensin receptor blockers protect podocytes more effectively than other anti-hypertensive drugs. Transgenic rats overexpressing angiotensin II Type 1 (AT1) receptor selectively in podocytes have been shown to develop glomerulosclerosis. The prevailing hypothesis is that angiotensin II has a capacity of directly acting on the AT1 receptor of podocytes to induce injury. We therefore investigated the mechanism of reno-protective effect of AT1 receptor in a mouse model of HIV-1 nephropathy. Methods We generated transgenic mice carrying the HIV-1 gene (control/HIV-1) or both HIV-1 gene and podocyte-selectively nullified AT1 gene (AT1KO/HIV-1). In these mice, we measured urinary protein or albumin excretion and performed histological analysis. Results At 8 months of age, AT1KO/HIV-1 (n = 13) and control/HIV-1 (n = 15) mice were statistically indistinguishable with respect to urinary albumin/creatinine ratio (median 2.5 versus 9.1 mg/mg), glomerulosclerosis (median 0.63 versus 0.45 on 0–4 scale) and downregulation of nephrin (median 6.90 versus 7.02 on 0–8 scale). In contrast to the observed lack of effect of podocyte-specific AT1KO, systemic AT1 inhibition with AT1 blocker (ARB) significantly attenuated proteinuria and glomerulosclerosis in HIV-1 mice. Conclusion These results indicate that the protective effect of ARB is mediated through its receptors on cells other than podocytes, such as efferent arteriolar smooth muscle cells. [ABSTRACT FROM AUTHOR]

Studies using lower organisms and cultured mammalian cells have revealed that the COP9 signalosome (CSN) has important roles in multiple cellular processes. Conditional gene targeting was recently used to study CSN function in murine T-cell development and activation. Using the Cre-loxP system, here we have achieved postnatal hepatocyte-restricted knockout of the csn8 gene (HR-Csn8KO) in mice. The protein abundance of other seven CSN subunits was differentially downregulated by HR-Csn8KO and the deneddylation of all cullins examined was significantly impaired. Moreover, HR-Csn8KO-induced massive hepatocyte apoptosis and evoked extensive reparative responses in the liver, including marked intralobular proliferation of biliary lineage cells and trans-differentiation and proliferation of the oval cells. However, division of pre-existing hepatocytes was significantly diminished in HR-Csn8KO livers. These findings indicate that Csn8 is essential to the ability of mature hepatocytes to proliferate effectively in response to hepatic injury. The histopathological examinations revealed striking hepatocytomegaly in Csn8-deficient livers. The hepatocyte nuclei were dramatically enlarged and pleomorphic with hyperchromasia and prominent nucleoli, consistent with dysplasia or preneoplastic cellular alteration in HR-Csn8KO mice at 6 weeks. Pericellular and perisinusoid fibrosis with distorted architecture was also evident at 6 weeks. It is concluded that CSN8/CSN is essential to postnatal hepatocyte survival and effective proliferation. [ABSTRACT FROM AUTHOR]

Puberty onset is initiated by activation of neurons that secrete gonadotropin-releasing hormone (GnRH). The timing and progression of puberty may depend upon temporal coordination of two opposing central mechanisms-a restraint of GnRH secretion before puberty onset, followed by enhanced stimulation of GnRH release to complete reproductive maturation during puberty. Neuronal estrogen receptor a (ERα) has been implicated in both controls; however, the underlying neural circuits are not well understood. Here we test whether these mechanisms are mediated by neurons that express kisspeptin, a neuropeptide that modulates GnRH neurosecretion. Strikingly, conditional ablation of ERα in kisspeptin neurons results in a dramatic advancement of puberty onset in female mice. Furthermore, subsequent pubertal maturation is arrested in these animals, as they fail to acquire normal ovulatory cyclicity. We show that the temporal coordination of juvenile restraint and subsequent pubertal activation is likely mediated by ERα in two separate kisspeptin neuronal populations in the hypothalamus. [ABSTRACT FROM AUTHOR]

Trophoblast invasion likely depends on complex cross talk between the fetal and maternal tissues and may involve the modulation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling activity in maternal decidual cells. In this report, we studied implantation in Ptentm1Hwu/tm1Hwu;Amhr2tm3(cre)Bhr/+ mice which lack the PI3K signaling antagonist gene Pten in myometrial and stromal/decidual cells. Primiparous Ptentm1Hwu/tm1Hwu;Amhr2tm3(cre)Bhr/+ mice were found to be subfertile because of increased fetal mortality at e11.5. Histopathological analyses revealed a failure of decidual regression in these mice, accompanied by reduced or absent invasion of fetal trophoblast glycogen cells and giant cells, abnormal development of the placental labyrinth, and frequent apparent intrauterine fetal growth restriction. Unexpectedly, the loss of phosphate and tensin homolog deleted on chromosome 10 (PTEN) expression in Ptentm1Hwu/tm1Hwu;Amhr2tm3(cre)Bhr/+ decidual cells was not accompanied by a detectable increase in AKT phosphorylation or altered expression or activation of PI3K/AKT downstream effectors such as mammalian target of rapamycin or glycogen synthase kinase-3β. Terminal deoxynucleotidyl transferase-mediated nick end labeling and bromodeoxyuridine incorporation analyses attributed to the lack of decidual regression mainly to decreased apoptosis in Ptentml Hwu/tm IHwu;Amhr2tm3(cre)Bhr/+ decidual cells, rather than to increased proliferation. Remodeling of the maternal vasculature was delayed in Ptentml Hw,~/tm IHwU;Amhr2tm3(cre)Bhr/+ uteri at el 1.5, as evidenced by persistence of vascular smooth muscle and decreased infiltration of uterine natural killer cells. In addition, thickening of the myometrium and disorganization of the muscle fibers were observed before and throughout gestation. Almost all Ptentmtm~H~~Amhr2tm3(cre)Bhr/+ mice failed to carry a second litter to term, apparently attributable to endometrial hyperplasia and uterine infections. Together, these data demonstrate novel roles of PTEN in the mammalian uterus and its requirement for proper trophoblast invasion and decidual regression. [ABSTRACT FROM AUTHOR]

To study the role of WNT4 in the postnatal ovary, a mouse strain bearing a floxed Wnt4 allele was created and mated to the Amhr2tm3(cre)Bhr strain to target deletion of Wnt4 to granulosa cells. Wnt4flox/-; Amhr2tm3(cre)Bhr/+ mice had reduced ovary weights and produced smaller litters (P<0.05). Serial follicle counting demonstrated that Wnt4fflox/-;Amhr2tm3(cre)Bhr/+ mice were born with a normal ovarian reserve and maintained normal numbers of small follicles until puberty but had only 25.2% of the normal number of healthy antral follicles. Some Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice had no antral follicles or corpora lutea and underwent premature follicle depletion. RT-PCR analyses of Wnt4flox/-; Amhr2tm3(cre)Bhr/+ granulosa cells and cultured granulosa cells that overexpress WNT4 demonstrated that WNT4 regulates the expression of Star, Cyp11a1, and Cyp19, steroidogenic genes previously identified as downstream targets of the WNT signaling effector CTNNB1. Decreased serum progesterone levels were found in immature, gonadotropin-treated Wnt4flox/-;Amhr2tm3(cre)Bhr/+ mice (P<0.05). WNT4- and CTNNB1-overexpressing cultured granulosa cells were analyzed by microarray for alterations in gene expression, which showed that WNT4 regulates additional genes involved in late follicle development via the WNT/CTNNB1 signaling pathway. Together, these data indicate that WNT4 is required for normal antral follicle development and may act by regulating granulosa cell functions including steroidogenesis. [ABSTRACT FROM AUTHOR]

The article presents the findings of the study on the role of myocardial G protein-coupled receptor kinase (GRK)2, which is a critical regulator of cardiac ß-adrenergic receptor (ßAR) signaling and cardiac function. It reveals that its upregulation in heart defect may continue to depress cardiac function and contribute to mortality in such syndrome. Moreover, the results establish a specific causal role for GRK2 in post-infarction cardiac remodeling.

Abstract: To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 ‘floxed’ mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo. [Copyright &y& Elsevier]

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that is widely expressed in the brain, and plays key roles in various cellular processes in response to both extracellular and intracellular stimuli. Here, we explored the role of FAK in cerebellar development. In the mouse cerebellum, FAK was found to be distributed as tiny cytoplasmic aggregates in various neuronal and glial elements, including Purkinje cells (PCs), Bergmann glia (BG), parallel fiber (PF)-terminals and climbing fiber (CF)-terminals. The neuron/glia-specific ablation of FAK impaired cerebellar foliation, such as variable decreases in foliation sizes and the lack of intercrural and precentral fissures. Some of the BG cells became situated ectopically in the molecular layer. Furthermore, the FAK ablation altered the innervation territories of CFs and PFs on PCs. CF innervation regressed to the basal portion of proximal dendrites and somata, whereas ectopic spines protruded from proximal dendrites and PFs expanded their territory by innervating the ectopic spines. Furthermore, the persistence of surplus CFs innervating PC somata caused multiple innervation. When FAK was selectively ablated in PCs, diminished dendritic innervation and persistent somatic innervation by CFs were observed, whereas cerebellar foliation and cell positioning of BG were normally retained. These results suggest that FAK in various neuronal and glial elements is required for the formation of normal histoarchitecture and cytoarchitecture in the cerebellum, and for the construction of proper innervation territory and synaptic wiring in PCs. [ABSTRACT FROM AUTHOR]

The apoptotic adapter protein FADD has been shown to play diverse roles in cell survival and proliferation. FADD knockout embryos died of heart defects, rendering Cre/ loxP-mediated conditional FADD knockout mice a unique tool for investigating FADD-dependent nonapoptotic mechanism. Previously, these genetically engineered mice were identified by time-consuming Southern blot or controversial real-time PCR. In this article, we report a novel genotyping strategy based on allele-specific inverse PCR (ASI-PCR) for rapid and reliable identification of conditional FADD knockout mice. In this strategy, the knockout nature of FADD was simply identified by screening the absence of the wild type FADD-specific ASI-PCR product. Using this method, we accurately identified CD4-Cre-mediated T cell specific FADD knockout mice. The whole process can be accomplished in any normal biological laboratory within 12 h using genomic DNA from tail biopsy. The proposed ASI-PCR-based approach is simple, rapid, sensitive, reproducible, and especially suitable for genotyping small amount of spatiotemporally restricted biopsies and large animal population. We believe that the strategy described in this article may be of general utility in genotyping other conditional gene knockout mice. [ABSTRACT FROM AUTHOR]

Abstract: In a first step towards dissecting molecular mechanisms that contribute to the development of cardiac diseases, we have generated transgenic mice that express a Cre–GFP fusion protein under the transcriptional control of a 4.3kb murine cardiac Troponin I gene (cTnI) promoter. Cre–GFP expression, similar in three transgenic lines, is described in one line. In mouse embryos, transgenic for the Cre–GFP and ROSA lacZ reporter allele, first Cre-mediated recombination appeared at 16.5 dpc selectively at the heart. Like the endogenous cTnI gene, transgenic Cre expression showed a slow rise through fetal development that increased neonatally. Bitransgenic hearts, stained at 30 days of age, showed intense signals in ventricular and atrial myocytes while no recombination occurred in other tissues. The delayed onset of Cre activity in cTnI–Cre mice could provide a useful genetic tool to evaluate the function of loxP targeted cardiac genes without interference of recombination during early heart development. [Copyright &y& Elsevier]

Abstract: Brain functions are the products of dynamic interactions between multiple genes and environments. Accordingly, there are large differences among mouse strains at the behavioral and neurobiological levels. Therefore, it is crucial to manipulate genes on the same and homogenous genetic background and then to analyze and compare the phenotypes of various genetically modified mice. Furthermore, a conditional gene targeting to restrict the gene knockout to specific cells and time is a powerful tool to investigate the molecular basis of higher brain functions such as learning and memory. We have developed a system employing Cre-progesterone receptor fusion recombinase for temporal regulation of gene targeting and Flp/frt recombination system for elimination of marker genes. Importantly, both the recombinase lines and target mice have been produced with embryonic stem cells derived from the C57BL/6 strain suitable for brain function analysis. Thus, we have established an inducible and neuron-specific gene targeting system on the pure C57BL/6 genetic background. [Copyright &y& Elsevier]

Abstract: Although the gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development, mice with a global inactivation of Cx43 survive until birth but die perinatally due to an obstruction of the right ventricular outflow tract of the heart. To analyze the functional role of Cx43 gap junction channels in cardiomyocytes of the developing and early postnatal heart, we used αMyHC-Cre mice to ablate Cx43 expression selectively in cardiomyocytes during development. We found efficient ablation of Cx43 in cardiomyocytes during embryonic development starting at embryonic day (ED) 9.5 in the ventricular wall. Analyses of cardiac Cx43 protein at birth indicated complete loss of Cx43 expression in cardiomyocytes. All mice homozygously deficient for Cx43 in cardiomyocytes died until postnatal day (PD) 16. Heterozygous inactivation of Cx43 in cardiomyocytes neither altered atrial nor ventricular activation, but homozygous ablation led to changes in ventricular activation, i.e. significant decrease of the QRS-amplitude and prolonged QRS-duration already at PD 4. Cardiac morphology was similar to controls until PD 1, but subtle morphological changes were found in a subgroup of mutant mice at later stages. Besides narrowing of the ventricular outlet region at PD 6, hypertrophy of ventricular myocardium was found at PD 12. Our data indicate that complete inactivation of cardiac Cx43 during development predisposes hearts to develop postnatal morphological alterations, which differ from outflow tract obstructions described for Cx43 null mice. In addition, complete loss of cardiac Cx43 protein during development correlates with slowed ventricular activation at PD 4, impairs viability during development, and leads to death of all mutant mice until PD 16. [Copyright &y& Elsevier]

Conditional gene targeting technique, which is based on the use of Cre-loxP or Flp/FRT systems, has been increasingly used to study gene function of a particular cell type in vivo. The introduction of this technique to the kidney field is relatively recent but has already provided important insights into physiological or pathological functions of a number of genes in the kidney. This technique has recently been used to inactivate the peroxisome proliferator-activated receptor subtype γ in the collecting duct, which leads to remarkable blockade of body weight gains and plasma volume expansion associated with thiazolidinediones. This finding not only helps understand pharmacology of the novel class of antidiabetic drugs, but also uncovers an important role of peroxisome proliferator-activated receptor subtype γ in regulation of distal nephron fluid reabsorption. The present review represents an example for the use of the modern technique to address complex clinical problems. It is anticipated that over next few years this technique will be used by an increasing number of investigators for studying gene function in the kidney. [ABSTRACT FROM AUTHOR]

Abstract: Notch genes encode evolutionarily conserved large, single transmembrane receptors, which regulate many cell fate decisions and differentiation processes during fetal and postnatal life. Multiple Notch receptors and ligands are expressed in both developing and adult epidermis and hair follicles. Proliferation and differentiation of these two ectodermal-derived structures have been proposed to be controlled in part by the Notch pathway. Whether Notch signaling is involved in postnatal hair homeostasis is currently unknown. Here, we investigate and compare the role of the Notch1 receptor during embryonic hair follicle development and postnatal hair homeostasis using Cre-loxP based tissue specific and inducible loss-of-function approaches. During embryonic development, tissue-specific ablation of Notch1 does not perturb formation and patterning of hair follicle placodes. However, Notch1 deficient hair follicles invaginate prematurely into the dermis. Embryonic as well as postnatal inactivation of Notch1 shortly after birth or in adult mice results in almost complete hair loss followed by cyst formation. The first hair cycle of Notch1 deficient mice is characterized by shortened anagen and a premature entry into catagen. These data show that Notch1 is essential for late stages of hair follicle development during embryogenesis as well as for post-natal hair follicle development and hair homeostasis. [Copyright &y& Elsevier]

Precise topological matching of presynaptic and postsynaptic specializations is essential for efficient synaptic transmission. Furthermore, synaptic connections are subjected to rearrangements throughout life. Here we examined the role of glutamate receptor (GluR) δ2 in the adult brain by inducible and cerebellar Purkinje cell (PC)-specific gene targeting under the pure C57BL/6 genetic background. Concomitant with the decrease of postsynaptic GluRδ2 proteins, presynaptic active zones shrank progressively and postsynaptic density (PSD) expanded, resulting in mismatching between presynaptic and postsynaptic specializations at parallel fiber-PC synapses. Furthermore, GluRδ2 and PSD-93 proteins were concentrated at the contacted portion of mismatched synapses, whereas AMPA receptors were distributed in both the contacted and dissociated portions. When GluRδ2 proteins were diminished, PC spines lost their synaptic contacts. We thus identified postsynaptic GluRδ2 as a key regulator of the presynaptic active zone and PSD organization at parallel fiber-PC synapses in the adult brain. [ABSTRACT FROM AUTHOR]

Previous studies have shown that Oct4 has an essential role in maintaining pluripotency of cells of the inner cell mass (ICM) and embryonic stem cells. However, Oct4 null homozygous embryos die around the time of implantation, thus precluding further analysis of gene function during development. We have used the conditional Cre/loxP gene targeting strategy to assess Oct4 function in primordial germ cells (PGCs). Loss of Oct4 function leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage, as has been described for Oct4-deficient ICM cells. These new results suggest a previously unknown function of Oct4 in maintaining viability of mammalian germline. [ABSTRACT FROM AUTHOR]

Sex determination in mammals directs an initially bipotential gonad to differentiate into either a testis or an ovary. This decision is triggered by the expression of the sex-determining gene Sry, which leads to the activation of male-specific genes including the HMG-box containing gene Sox9. From transgenic studies in mice it is clear that Sox9 is sufficient to induce testis formation. However, there is no direct confirmation for an essential role for Sox9 in testis determination. The studies presented here are the first experimental proof for an essential role for Sox9 in mediating a switch from the ovarian pathway to the testicular pathway. Using conditional gene targeting, we show that homozygous deletion of Sox9 in XY gonads interferes with sex cord development and the activation of the male-specific markers Mis and P450scc, and leads to the expression of the female-specific markers Bmp2 and follistatin. Moreover, using a tissue specific knock-out approach, we show that Sox9 is involved in Sertoli cell differentiation, the activation of Mis and Sox8, and the inactivation of Sry. Finally, double knock-out analyses suggest that Sox8 reinforces Sox9 function in testis differentiation of mice. [ABSTRACT FROM AUTHOR]

The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43–/–) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types. [Copyright &y& Elsevier]

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background. [Copyright &y& Elsevier]

To elucidate the biological role of Stat3 in the skin, conditional gene targeting using the Cre-loxP system was performed as germline Stat3 ablation leads to embryonic lethality. K5-Cre;Stat3flox/-transgenic mice, whose epidermal and follicular keratinocytes lack functional Stat3, were viable and the development of epidermis and hair follicles appeared normal. However, hair cycle and wound healing processes were severely compromised. Furthermore, mutant mice expressed sparse hair and developed spontaneously occurring ulcers with age. Growth factor-dependent in vitro migration of Stat3-disrupted keratinocytes was impaired despite normal proliferative responses. We therefore conclude that Stat3 plays a crucial role in transducing a signal required for migration but not for proliferation of keratinocytes, and that Stat3 is essential for skin remodeling, including hair cycle and wound healing. [ABSTRACT FROM AUTHOR]

Over the past two decades the techniques associated with the manipulation of the mouse genome have provided a powerful approach toward the better understanding of gene function. Conventional transgenie and gene targeting approaches have been used extensively, and these techniques have been particularly rewarding for neuroscientists. Nevertheless, the traditional approaches toward genome manipulation have certain limitations that diminish their usefulness for studying more sophisticated biological processes. Therefore, variations to these techniques have recently been developed. The improvements are focused on two areas: one provides regulated control of transgene expression using an inducible expression system; and the other provides the opportunity to inactivate genes in specific cells and at predetermined developmental stages with a conditional gene targeting system. This review summarizes the advantages as well as some of the technical difficulties of these new approaches. The application of these advanced approaches in biomedical research, particularly neuroscience, are also discussed. [ABSTRACT FROM AUTHOR]

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Interleukin-10 (IL-10) is a potent immunomodulatory cytokine, whose cellular targets have not yet been precisely identified. Dendritic cell (DC)-specific IL-10 receptor knockout mice exhibit exaggerated T-cell reactivation in the skin, highlighting a key role of DCs in the maintenance of local immune homeostasis, beyond their classical function as regulators of T-cell priming in lymph nodes. [ABSTRACT FROM AUTHOR]