A Pig-a conditional knock-out mice model mediated by Vav-iCre: stable GPI-deficient and mild hemolysis

Paroxysmal nocturnal hemoglobinuria is a clonal disease caused by PIG-A mutation of hematopoietic stem cells. At present, there is no suitable PNH animal model for basic research, therefore, it is urgent to establish a stable animal model. We constructed a Pig-a conditional knock-out mice model by ES targeting technique and Vav-iCre. The expressions of GPI and GPI-AP were almost completely absent in CKO homozygote mice, and the proportion of the deficiency remained stable from birth. In CKO heterozygote mice, the proportion of the deficiency of GPI and GPI-AP was partially absent and decreased gradually from birth until it reached a stable level at 3 months after birth and remained there for life. Compared with normal C57BL/6N mice and Flox mice, pancytopenia was found in CKO homozygous mice, and leukopenia and anemia were found in CKO heterozygotes mice. Meanwhile, in CKO mice, the serum LDH, TBIL, IBIL, complement C5b-9 levels were increased, and the concentration of plasma FHb was increased. Hemosiderin granulosa cells can be seen more easily in the spleens of CKO mice. What’s more, CKO mice had stable transcription characteristics. In conclusion, our mouse model has stable GPI-deficient and mild hemolysis, which may be an ideal in vivo experimental model for PNH.

Endothelial Tyrosine Kinase Tie1 Is Required for Normal Schlemm’s Canal Development

Background: Schlemm’s canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. Methods: We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. Results: Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. Conclusions: Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

Prolonged nicotine exposure reduces aversion to the drug in mice by altering nicotinic transmission in the interpeduncular nucleus.

Nicotine intake is likely to result from a balance between the rewarding and aversive properties of the drug, yet the individual differences in neural activity that control aversion to nicotine and their adaptation during the addiction process remain largely unknown. Using a two-bottle choice experiment, we observed a high heterogeneity in nicotine-drinking profiles in isogenic adult male mice, with about half of the mice persisting in consuming nicotine even at high concentrations, whereas the other half durably stopped consumption. We found that nicotine intake was negatively correlated with nicotine-evoked currents in the interpeduncular nucleus (IPN), and that prolonged exposure to nicotine, by weakening this response, decreased aversion to the drug, and hence boosted consumption. Lastly, using knock-out mice and local gene re-expression, we causally identified β4-containing nicotinic acetylcholine receptors of IPN neurons as the molecular and cellular correlates of nicotine aversion. Collectively, our results identify the IPN as a substrate of individual variabilities and adaptations in nicotine consumption.

Dopamine D2Rs Coordinate Cue-Evoked Changes in Striatal Acetylcholine Levels

In the striatum, acetylcholine (ACh) neuron activity is modulated co-incident with dopamine (DA) release in response to unpredicted rewards and reward predicting cues and both neuromodulators are thought to regulate each other. While this co-regulation has been studied using stimulation studies, the existence of this mutual regulation in vivo during natural behavior is still largely unexplored. One long-standing controversy has been whether striatal DA is responsible for the induction of the cholinergic pause or whether D2R modulate a pause that is induced by other mechanisms. Here, we used genetically encoded sensors in combination with pharmacological and genetic inactivation of D2Rs from cholinergic interneurons (CINs) to simultaneously measure ACh and DA levels after CIN D2R inactivation. We found that CIN D2Rs are not necessary for the induction of cue induced dips in ACh levels but regulate dip lengths and rebound ACh levels. Importantly, D2R inactivation strongly decreased the temporal correlation between DA and Ach signals not only at cue presentation but also during the intertrial interval. This points to a general mechanism by which D2Rs coordinate both signals. At the behavioral level D2R antagonism increased the latency to lever press, which was not observed in CIN-selective D2R knock out mice. This latency correlated with the cue evoked dip length supporting a role of the ACh dip and it’s regulation by D2Rs in motivated behavior. Overall, our data indicate that striatal DA coordinate phasic ACh and DA signals via CIN D2Rs which is important for the regulation of motivated behavior.

RB depletion is required for the continuous growth of tumors initiated by loss of RB

The retinoblastoma (RB) tumor suppressor is functionally inactivated in a wide range of human tumors where this inactivation promotes tumorigenesis in part by allowing uncontrolled proliferation. RB has been extensively studied, but its mechanisms of action in normal and cancer cells remain only partly understood. Here, we describe a new mouse model to investigate the consequences of RB depletion and its re-activation in vivo. In these mice, induction of shRNA molecules targeting RB for knock-down results in the development of phenotypes similar to Rb knock-out mice, including the development of pituitary and thyroid tumors. Re-expression of RB leads to cell cycle arrest in cancer cells and repression of transcriptional programs driven by E2F activity. Thus, continuous RB loss is required for the maintenance of tumor phenotypes initiated by loss of RB, and this new mouse model will provide a new platform to investigate RB function in vivo.

Isolation-Induced Ultrasonic Vocalization in Environmental and Genetic Mice Models of Autism

Studies in rodent models suggest that calls emitted by isolated pups serve as an early behavioral manifestation of communication deficits and autistic like behavior. Previous studies in our labs showed that gestational exposure to the pesticide chlorpyrifos (CPF) and the Mthfr-knock-out mice are associated with impaired social preference and restricted or repetitive behavior. To extend these studies, we examine how pup communication via ultrasonic vocalizations is altered in these ASD models. We implemented an unsupervised hierarchical clustering method based on the spectral properties of the syllables in order to exploit syllable classification to homogeneous categories while avoiding over-categorization. Comparative exploration of the spectral and temporal aspects of syllables emitted by pups in two ASD models point to the following: (1) Most clusters showed a significant effect of the ASD factor on the start and end frequencies and bandwidth and (2) The highest percent change due to the ASD factor was on the bandwidth and duration. In addition, we found sex differences in the spectral and temporal properties of the calls in both control groups as well as an interaction between sex and the gene/environment factor. Considering the basal differences in the characteristics of syllables emitted by pups of the C57Bl/6 and Balb/c strains used as a background in the two models, we suggest that the above spectral-temporal parameters start frequency, bandwidth, and duration are the most sensitive USV features that may represent developmental changes in ASD models.

In Vitro Folliculogenesis in Mammalian Models: A Computational Biology Study

In vitro folliculogenesis (ivF) has been proposed as an emerging technology to support follicle growth and oocyte development. It holds a great deal of attraction from preserving human fertility to improving animal reproductive biotechnology. Despite the mice model, where live offspring have been achieved,in medium-sized mammals, ivF has not been validated yet. Thus, the employment of a network theory approach has been proposed for interpreting the large amount of ivF information collected to date in different mammalian models in order to identify the controllers of the in vitro system. The WoS-derived data generated a scale-free network, easily navigable including 641 nodes and 2089 links. A limited number of controllers (7.2%) are responsible for network robustness by preserving it against random damage. The network nodes were stratified in a coherent biological manner on three layers: the input was composed of systemic hormones and somatic-oocyte paracrine factors; the intermediate one recognized mainly key signaling molecules such as PI3K, KL, JAK-STAT, SMAD4, and cAMP; and the output layer molecules were related to functional ivF endpoints such as the FSH receptor and steroidogenesis. Notably, the phenotypes of knock-out mice previously developed for hub.BN indirectly corroborate their biological relevance in early folliculogenesis. Finally, taking advantage of the STRING analysis approach, further controllers belonging to the metabolic axis backbone were identified, such as mTOR/FOXO, FOXO3/SIRT1, and VEGF, which have been poorly considered in ivF to date. Overall, this in silico study identifies new metabolic sensor molecules controlling ivF serving as a basis for designing innovative diagnostic and treatment methods to preserve female fertility.