New design of a lentiviral shRNA vector with inducible co-expression of ARGONAUTE 2 for enhancing gene silencing efficiency

Background: RNA interference (RNAi) is a robust tool for inhibiting specific gene expression, but it is limited by the uncertain efficiency of siRNA or shRNA constructs. It has been shown that the overexpression of ARGONAUTE 2 (AGO2) protein increases silencing efficiency. However, the key elements required for AGO2-mediated enhancement of gene silencing in lentiviral vector has not been well studied.Results: To explore the application of AGO2-based shRNA system in mammalian cells, we designed shRNA vectors targeting the EGFP reporter gene and evaluated the effects of various factors on silencing efficiency including stem length, loop sequence, antisense location as well as the ratio between AGO2 and shRNA. We found that 19 ~ 21-bp stem and 6- or 9-nt loop structure in the sense-loop-antisense (S-L-AS) orientation was an optimal design in the AGO2-shRNA system. Then, we constructed a single lentiviral vector co-expressing shRNA and AGO2 and demonstrated that the simultaneous expression of shRNA and AGO2 can achieve robust silencing of exogenous DsRed2 and endogenous ID1 and P65 genes. However, the titers of packaged lentivirus from constitutive expression of AGO2 vector were extremely low, severely limiting its broad application. For the first time, we demonstrated that the problem can be significantly improved by using the inducible expression of AGO2 lentiviral system.Conclusions: We reported a novel lentiviral vector with an optimal design of shRNA and inducible AGO2 overexpression which provides a new tool for RNAi research.Peer reviewedPhysiological Science

Opposite effects of MK-801 on the expression of food and morphine-induced conditioned place preference in rats

Behavioural studies have provided strong evidence for common substrates in the rewards of natural and addictive substances, but it is still unclear whether there is a common glutamatergic NMDA receptor mechanism involved in the processing of reward for both. The present study was designed to investigate the effects of MK-801 (0.1mg/kg) on the expression of place preference conditioned with food and morphine (5.0mg/kg) in rats. The data indicates that MK-801 potentiates the expression of food-induced conditioned place preference (CPP) but retards that of morphine CPP. It also demonstrates that the opposite effects of MK-801 on food and morphine CPP expression were caused neither by hyperactivity nor by the impairment of memory retrieval. These results suggest that MK-801 enhances food craving and inhibits morphine craving in rats, and that the roles of glutamatergic NMDA receptor mechanisms in the reward processing of natural reinforcers and addictive drugs may be dissociable.Behavioural studies have provided strong evidence for common substrates in the rewards of natural and addictive substances, but it is still unclear whether there is a common glutamatergic NMDA receptor mechanism involved in the processing of reward for both. The present study was designed to investigate the effects of MK-801 (0.1 mg/kg) on the expression of place preference conditioned with food and morphine (5.0 mg/kg) in rats. The data indicates that MK-801 potentiates the expression of food-induced conditioned place preference (CPP) but retards that of morphine CPP. It also demonstrates that the opposite effects of MK-801 on food and morphine CPP expression were caused neither by hyperactivity nor by the impairment of memory retrieval. These results suggest that MK-801 enhances food craving and inhibits morphine craving in rats, and that the roles of glutamatergic NMDA receptor mechanisms in the reward processing of natural reinforcers and addictive drugs may be dissociable

The ability of acute corticosterone to facilitate morphine-induced conditioned locomotion activity can be dissociated from its effects on conditioned place preference

There is evidence that the biological mechanism of psychomotor stimulant properties is the same as that of rewarding effects of addictive drugs. However, whether this relationship also exists between conditioned place preference and conditioned locomotion activity remains unclear. Male SD rats were investigated the effect of acute corticosterone on morphine-induced CPP and conditioned locomotion activity. It indicated that corticosterone increased acute psychomotor activity and conditioned psychomotor activity of morphine-treated rats, but inhibited the acquisition of morphine-induced CPP, The results suggested that the biological mechanism of CPP may be different from, at least under acute stress, that of conditioned locomotor activity

Novelty-seeking behavior and stress-induced locomotion in rats of juvenile period differentially related to morphine place conditioning in their adulthood

The relationship between motor responses in a novel environment and susceptibility to place conditioning effect of psychostimulants has been reported in adult rats. However, it is in question whether this correlation could be generalized to motor activity in rats of juvenile period and place conditioning effect in their adulthood for narcotic morphine. In the present study, we tested locomotor activity in an arena open-field and the subsequent novelty-seeking behavior after adaptation process in juvenile rats (P42) and morphine (2 mg/kg) place conditioning effect 56 days later in the same rats' adulthood (P98). Our results showed that rats with high response to novelty (HRN) spent more prolonged duration in the drug-paired compartment in the place conditioning test compared with their low response counterparts (LRN), with the latter group no salient change on this measure. Moreover, rats with high response to the open-field test (HRS) expressed equally elevated duration in drug-paired side relative to their low response counterparts (LRS). The present research demonstrated that novelty-seeking behavior and locomotor activity in the open-field in rats of juvenile period differentially related to morphine place conditioning in their adulthood, with slow acquisition of morphine place conditioning effect in LRN animalsThe relationship between motor responses in a novel environment and susceptibility to place conditioning effect of psychostimulants has been reported in adult rats. However, it is in question whether this correlation could be generalized to motor activity in rats of juvenile period and place conditioning effect in their adulthood for narcotic morphine. In the present study, we tested locomotor activity in an arena open-field and the subsequent novelty-seeking behavior after adaptation process in juvenile rats (P42) and morphine (2 mg/kg) place conditioning effect 56 days later in the same rats' adulthood (P98). Our results showed that rats with high response to novelty (HRN) spent more prolonged duration in the drug-paired compartment in the place conditioning test compared with their low response counterparts (LRN), with the latter group no salient change on this measure. Moreover, rats with high response to the open-field test (HRS) expressed equally elevated duration in drug-paired side relative to their low response counterparts (LRS). The present research demonstrated that novelty-seeking behavior and locomotor activity in the open-field in rats of juvenile period differentially related to morphine place conditioning in their adulthood, with slow acquisition of morphine place conditioning effect in LRN animals. (C) 2003 Elsevier B.V. All rights reserved

Effect of corticosterone on morphine-induced psychomotor effect and its sensitization

Previous research demonstrated important roles of stress in behavioral effects of drug abuse. However, whether extraneous stress factors could differentially modulate morphine psychomotor effect and its sensitization is still unknown. This study examined the influence of corticosterone on the acute effect of morphine and its sensitization. Morphine (2 mg.kg-1 i.p.) enhanced the locomotor activity and formed behavioral sensitization upon repeated exposure. Corticosterone (5 mg.kg-1 i.p.) increased morphine psychomotor effect but played null role in the modulation of its sensitization. These results indicated that stress exposure promoted morphine acute psychomotor effect but did not affect the development of morphine sensitization

Effects of scopolamine and physostigmine on acquisition of morphine-treated rats in Morris water maze performance

AIM: To investigate effects of morphine on acquisition process of rats a nd interactions of opioid and cholinergic systems by Morris water maze performance.METHODS: Morris water maze was used to measure the latency of rats with drug s treatment to find the covert platform.RESULTS: Chronic morphine administration (10 mg/kg) impaired the acquisition process of rats in Morris water maze task. Appreciable difference was identified with morphine 10 mg/k g group compared with morphine 3 mg/kg group. Co-administration of morphine (10 mg/kg) and scopolamine (3 mg/kg) aggravated acquisition impairment induced by morphine 1 0 mg/kg or scopolamine alone, though scopolamine itself induced no salient changes in acquisition capabilities of rats. In addition, physostigmine (0.1 mg/kg) could appreciably attenuate morphine-induced acquisition impairment.CONCLUSION: Morphine 10 mg/kg evidently impaired acquisition process of rats. There was a close relationship between the acquisition capabilities of morphine-treated rats and the functions of cholinergic system.AIM: To investigate effects of morphine on acquisition process of rats and interactions of opioid and cholinergic systems by Morris water maze performance. METHODS: Morris water maze was used to measure the latency of ruts With drugs treatment to find the covert platform. RESULTS: Chronic morphine administration (10 mg/kg) impaired the acquisition process of rats in Morris water maze task. Appreciable difference was identified With morphine 10 mg/kg group compared with morphine 3 mg/kg group. Co-administration of morphine ( 10 mg/kg) and scopolamine (3 mg/kg) aggravated acquisition impairment induced by morphine 10 mg/kg or scopolamine alone, though scopolamine itself induced no salient changes in acquisition capabilities of rats. In addition, physostigmine (0.1 mg/kg) could appreciably attenuate morphine-induced acquisition impairment. CONCLUSION: Morphine 10 mg/kg evidently impaired acquisition process of rats. There was a close relationship between the acquisition capabilities of morphine-treated rats and the functions of cholinergic system

Susceptibility to morphine place conditioning: relationship with stress-induced locomotion and novelty-seeking behavior in juvenile and adult rats

Previous studies demonstrated that the rewarding effect of psychostimulants, such as amphetamine and cocaine, can be predicted by locomotor activity toward novelty in a free-choice situation but not motor response developed in inescapable environment. However, whether this relationship also exists with narcotic morphine remains unclear. In the present study, the relationship between morphine place conditioning and open field as well as novelty-seeking behavior was examined in both juvenile and adult rats. By using arena open field and the same arena containing novel object, we investigated the initial open-field activity and novelty-seeking behavior after familiarization process, respectively, in juvenile and adult rats. Subsequently, the relationship between morphine (2 mg/kg) place conditioning and the above two behaviors was examined. Our results demonstrated that morphine place conditioning effect was readily acquired in both groups. The magnitude of this effect positively correlated with novelty-seeking intensity but not with open-field activity. This is the case whether juvenile or adult group was examined separately or across ages. However, only rats with high response to novelty (NHR) from their respective group expressed significant duration increase in drug-paired compartment. Rats with low response to novelty (NLR) showed no sign of this effect after the same drug training, suggesting slow acquisition of this effect in NLRs. These results also indicated that novelty-seeking actions and the rewarding effect of morphine possessed a common pathway and that neural and hormonal substrates activated in a mild stress environment like in the open field may not be critically involved in this process. The ontogenetic specificity and nonspecificity between different-aged rats as with the above relationship were discussed in this paper.Previous studies demonstrated that the rewarding effect of psychostimulants, such as amphetamine and cocaine, can be predicted by locomotor activity toward novelty in a free-choice situation but not motor response developed in inescapable environment. However, whether this relationship also exists with narcotic morphine remains unclear. In the present study, the relationship between morphine place conditioning and open field as well as novelty-seeking behavior was examined in both juvenile and adult rats. By using arena open field and the same arena containing novel object, we investigated the initial open-field activity and novelty-seeking behavior after familiarization process, respectively, in juvenile and adult rats. Subsequently, the relationship between morphine (2 mg/kg) place conditioning and the above two behaviors was examined. Our results demonstrated that morphine place conditioning effect was readily acquired in both groups. The magnitude of this effect positively correlated with novelty-seeking intensity but not with open-field activity. This is the case whether juvenile or adult group was examined separately or across ages. However, only rats with high response to novelty (NHR) from their respective group expressed significant duration increase in drug-paired compartment. Rats with low response to novelty (NLR) showed no sign of this effect after the same drug training, suggesting slow acquisition of this effect in NLRs. These results also indicated that novelty-seeking actions and the rewarding effect of morphine possessed a common pathway and that neural and hormonal substrates activated in a mild stress environment like in the open field may not be critically involved in this process. The ontogenetic specificity and nonspecificity between different-aged rats as with the above relationship were discussed in this paper. (C) 2003 Elsevier Inc. All rights reserved

Effect of dietary soybean lecithin on reproductive performance of Chinese mitten crab Eriocheir sinensis (H. Milne-Edwards) Broodstock

The effect of increasing levels of dietary phospholipids (PL) on the ovarian development and reproductive performance of Chinese mitten crab Eriocheir sinensis were investigated using four semipurified formulated diets supplemented with 0%, 1.2%, 2.4%, and 3.6% PL. Four groups of 40 females, with an average individual body weight of 95-120 g, were fed the experimental diets for a period of 7 months. Male crabs were introduced into the female rearing system in March, and mating, spawning, and egg hatching occurred in the following month. After 10 weeks of feeding, females fed the diet with 2.4% PL had a significantly higher gonadosomatic index (GSI) than females fed the diet with 0% PL, whereas females fed the diet with 1.2% PL had a significantly higher hepatic moisture content and lower hepatic lipid content than the other groups (P < 0.05). After mating, the spawning rate was higher among females fed diets with 3.6% and 2.4% PL (95% and 92%, respectively) compared with females fed diets with 0% and 1.2% PL (both 81%). The results showed that egg production (total number of eggs/female) and fecundity increased with increasing dietary PL level, with females fed diets with 0% and 1.2% PL supplementation having significantly lower values than females fed the diet with 3.6% PL (P < 0.05). In conclusion, our results suggest that dietary PL supplementation has a positive effect on ovarian development and reproductive performance of E. sinensis broodstock. Further study should aim to investigate the optimal PL level in the broodstock diet of E. sinensis in respect to offspring quality

Effect of dietary HUFA on tissue fatty acid composition and reproductive performance of Chinese mitten crab Eriocheir sinensis (H. Milne-Edwards) broodstock

This study was conducted to determine the optimum highly unsaturated fatty acid (HUFA) level in semi-purified diets for female Chinese mitten crab Eriocheir sinensis broodstock. Five isolipidic and isonitrogenous diets were formulated to contain different HUFA levels (0.14, 0.65, 1.15, 1.64, and 2.07%, respectively) by the supplementation of HUFA-rich fish oil. Diets were fed to triplicate groups of 25 female crabs (75 animals per treatment) stocked in concrete tanks for 8 months. The hepatopancreas, ovary, and muscle of the crabs were sampled after 3-month culturing, and the eggs were sampled after 8-month culturing. The reproductive performance of E. sinensis broodstock was evaluated in relation to the different dietary HUFA levels. Moreover, the fatty acid composition of the hepatopancreas, ovary, muscle, and eggs was determined in crabs fed various levels of HUFA. The results showed that a significant positive correlation was observed between the dietary HUFA level and the HUFA content in hepatopancreas, ovary, and egg, respectively. However, no significant difference in gonadosomatic index, hepatosomatic index, and total ovarian and hepatopancreatic lipid content among the treatments was observed after 3-month culturing. Also, the reproductive performance was similar for all treatments after 8-month culturing. The fact of lacking significant correlation between reproductive parameters and dietary HUFA level was discussed in respect of the catadromous life cycle of E. sinensis and warranted further research with more comprehensive approaches