BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high dose...BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high doses of drug are required and the success rate of model induction is low. It is necessary to develop an improved method to establish a temporal lobe epilepsy （TLE） animal model. OBJECTIVE： To explore an economic, stable and efficient method of establishing a TLE animal model. DESIGN, TIME AND SETTING： A completely randomized, controlled study. The experiments were performed in the Cellular Function Laboratory of the Physiology Department, Anhui Medical University from March to July 2007. MATERIALS： Twenty adult male Wistar rats, weighing 230-260 g, were provided by the Experimental Animal Centre of Nanjing Medical University. Kainic acid was purchased from Sigma in USA. Type SN-2 stereotaxic apparatus was made by Narishge in Japan. METHODS： Wistar rats were randomly divided into a kainic acid （KA） group （n = 12） and a normal saline （NS） group （n = 8）. For intrahippocampal microinjection, a burr hole was drilled in the skull at the following stereotaxic coordinates： anteroposterior （AP） 4.1 mm caudal to bregma; lateral （ML） 4.2 mm right lateral to the midline. Rats in the KA group were injected with 2.5 μL KA （0.4 g/L） into the center of the CA3 region, while in the NS group the same volume of NS was injected into the same site. MAIN OUTCOME MEASURES： Both groups were monitored under a video capture system for 12 weeks to record spontaneous seizures. Intracranial eletroencepholograph （IEEG） recordings in vivo were performed after the behavioral observations. After the IEEG recordings, hippocampi were processed into coronal sections. Nissl and Timm stainings were then performed to observe and confirm pathology. RESULTS： Twenty rats were involved in the final analysis. Behavioral observations： the eadiest spontaneous onset of epilepsy appeared 2 weeks after injection of KA. Eight rats had spontaneous onset of epilepsy 3-12 weeks after treatment. None of rats in the NS group had spontaneous onset of epilepsy. IEEG recordings： Epileptic-form waves, such as sharp waves and spike waves, were calculated by artificial analysis The number of epileptic-form waves in the KA group increased significantly compared to those of the NS group （P 〈 0.01）. Morphology results： In the KA group, Nissl staining and Timm staining revealed typical pathology in the hippocampal temporosphenoid lobe. In the NS group, no pathology was observed. CONCLUSION： Intrahippocampal microinjection of KA is a reliable method to establish a temporal lobe epilepsy animal model, requiring low doses of kainic acid and giving a high rate of success.展开更多
Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factor...Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.展开更多
The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we...The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.展开更多
Daidzein (DA) was microinjected into mediobasal hypothalamus (MBH) and nucleus ventrome-dialis thalami (VM) of the castrated male Goettingen Mini pigs (n = 9, 10 μl·animal-1, 8 pgDA·μl-1 and plasma luteini...Daidzein (DA) was microinjected into mediobasal hypothalamus (MBH) and nucleus ventrome-dialis thalami (VM) of the castrated male Goettingen Mini pigs (n = 9, 10 μl·animal-1, 8 pgDA·μl-1 and plasma luteinizing hormone (LH) levels were compared between the prior and the posterior treatment. The result showed that LH levels after the cerebral administration (ad) tended to increase compared to the levels before ad. In MBH, LH levels in 4 cases (4/5), rose and were not changed in 1 case at 0.5 - 2 hours after ad compared to those before ad. There were no significant changes at 2.5 hours after ad. When it was injected in VM, LH levels in 3 cases (3/4) rose, and were not changed in 1 case after ad compared to those before ad. In the control, there were no changes in plasma LH levels between the pre-and post-treatment except 1 case in MBH. This study suggested that DA could up-regulate LH secretion through hypothalamus level in the castrated boars.展开更多
Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard met...Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.展开更多
Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (onl...Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.展开更多
The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select b...The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse genetics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40%of larvae.The proportion of normal-hatched larvae was approximately 50%.Different mutations,including short indels and fragment deletions,were found in our test genes gsdf and myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.展开更多
Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods S...Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods Superovulation and synchronization of estrus were induced in female Japanese White Rabbits by injecting hormone, then mating with male. After collected the fertilized eggs, the human apoA-Ⅱ gene was microinjected into the male pronucleus of eggs. The injected eggs were transferred into recipient female rabbits. Last, extract DNA from the new borns ear and determine whether the newborns were transgenic by polymerase chain reaction (PCR) or Southern blot analysis. Results A total of 822 embryos with microinjection of human apoAⅡ gene were implanted into 28 recipient rabbits. The number of surviving newborns was 37. 3 transgenic positive surviving founders were found with human apoA-Ⅱ.展开更多
基金Funds for the Excellent Talent of Anhui Province of China, No.06043090National Century Excellent Talents in University of China, No.NCET-06-0557Natural Science Foundation of Anhui Province Department of Education, No. KJ2007A028
文摘BACKGROUND： Kainic acid can be used to induce a model of epilepsy by systemic injection, such as intraperitoneal or subcutaneous injection. Individual rats have different responses to kainic acid, therefore high doses of drug are required and the success rate of model induction is low. It is necessary to develop an improved method to establish a temporal lobe epilepsy （TLE） animal model. OBJECTIVE： To explore an economic, stable and efficient method of establishing a TLE animal model. DESIGN, TIME AND SETTING： A completely randomized, controlled study. The experiments were performed in the Cellular Function Laboratory of the Physiology Department, Anhui Medical University from March to July 2007. MATERIALS： Twenty adult male Wistar rats, weighing 230-260 g, were provided by the Experimental Animal Centre of Nanjing Medical University. Kainic acid was purchased from Sigma in USA. Type SN-2 stereotaxic apparatus was made by Narishge in Japan. METHODS： Wistar rats were randomly divided into a kainic acid （KA） group （n = 12） and a normal saline （NS） group （n = 8）. For intrahippocampal microinjection, a burr hole was drilled in the skull at the following stereotaxic coordinates： anteroposterior （AP） 4.1 mm caudal to bregma; lateral （ML） 4.2 mm right lateral to the midline. Rats in the KA group were injected with 2.5 μL KA （0.4 g/L） into the center of the CA3 region, while in the NS group the same volume of NS was injected into the same site. MAIN OUTCOME MEASURES： Both groups were monitored under a video capture system for 12 weeks to record spontaneous seizures. Intracranial eletroencepholograph （IEEG） recordings in vivo were performed after the behavioral observations. After the IEEG recordings, hippocampi were processed into coronal sections. Nissl and Timm stainings were then performed to observe and confirm pathology. RESULTS： Twenty rats were involved in the final analysis. Behavioral observations： the eadiest spontaneous onset of epilepsy appeared 2 weeks after injection of KA. Eight rats had spontaneous onset of epilepsy 3-12 weeks after treatment. None of rats in the NS group had spontaneous onset of epilepsy. IEEG recordings： Epileptic-form waves, such as sharp waves and spike waves, were calculated by artificial analysis The number of epileptic-form waves in the KA group increased significantly compared to those of the NS group （P 〈 0.01）. Morphology results： In the KA group, Nissl staining and Timm staining revealed typical pathology in the hippocampal temporosphenoid lobe. In the NS group, no pathology was observed. CONCLUSION： Intrahippocampal microinjection of KA is a reliable method to establish a temporal lobe epilepsy animal model, requiring low doses of kainic acid and giving a high rate of success.
基金supported by a grant from State Key Laboratory of Proteomics of China,No.SKLP-K201401(to SJL)the National Key Project of Basic Research of China,No.2009CB918301(to SJL)the National Natural Science Foundation of China,Nos.30430310,30140001,30370460(to SJL)
文摘Microglia, as the resident immune cells in the central nervous system, play important roles in regulating neuronal processes, such as neural excitability, synaptic activity, and apoptotic cell clearance. Growth factors can activate multiple signaling pathways in central nervous system microglia and can regulate their immune effects, but whether growth factors can affect the morphological characteristics and ultrastructure of microglia has not been reported. After microinjecting 300 nL of a growth factor cocktail, including 10 μg/mL epidermal growth factor, 10 μg/mL basic fibroblast growth factor, 10 μg/mL hepatocyte growth factor and 10 μg/mL insulin-like growth factor into adult rat cortex, we found that the number of IBA1-positive microglia around the injection area increased significantly, indicating local activation of microglia. All CD68-positive labeling co-localized with IBA1 in microglia. Cell bodies and protrusions of CD68-positive cells were strongly attached to or were engulfing neurons. Characteristic huge phagosomes were observed in activated phagocytes by electron microscopy. The phagosomes generally included non-degraded neuronal protrusions and mitochondria, yet they contained no myelin membrane or remnants, which might indicate selective phagocytosis by the phagocytes. The remnant myelin sheath after phagocytosis still had regenerative ability and formed "myelin-like" structures around phagocytes. These results show that microinjection of a growth factor cocktail into the cerebral cortex of rodents can locally activate microglia and induce selective phagocytosis of neural structures by phagocytes. The study was approved by the Institute of Laboratory Animal Science, Beijing Institute of Basic Medical Sciences(approval No. IACUC-AMMS-2014-501) on June 30, 2014.
基金This work was supported by the National Natural Science Foundation of China(32070502,31730074,32072419 and 31501905).
文摘The mutant efficiency and hatching ratio are two key factors that significantly affect the construction of genome-modified mutant insects.In the construction of CRISPR/Cas9-mediated dsLmRNase2^(–/–)mutant locusts,we found that the tanned eggs which experienced a 20-min contact with the oocyst exhibited a higher success rate compared to fresh newly-laid eggs that were less tanned.However,the heritable efficiency of the dsLmRNase2 deletion to the next generation G_(1 )progeny was similar between adults derived from the tanned or less tanned engineered eggs.Further,the similar effective mutant ratios in the normally developed eggs and G_(0) adults of tanned and less tanned eggs also indicated that tanning did not reduce the absolute mutation efficiency induced by CRISPR/Cas9.Moreover,we found that the syncytial division period,which was longer than the time for tanning,conferred a window period for microinjection treatment with efficient mutation in both tanned and less tanned eggs.We further found that tanned eggs exhibited a higher hatching rate due to a reduced infection rate following microinjection.Both the anti-pressure and ultrastructure analyses indicated that the tanned eggs contained compressed eggshells to withstand increased external pressure.In summary,tanned eggs possess stronger defense responses and higher efficiency of genome editing,providing an improved model for developing Cas9-mediated gene editing procedures in locusts.
文摘Daidzein (DA) was microinjected into mediobasal hypothalamus (MBH) and nucleus ventrome-dialis thalami (VM) of the castrated male Goettingen Mini pigs (n = 9, 10 μl·animal-1, 8 pgDA·μl-1 and plasma luteinizing hormone (LH) levels were compared between the prior and the posterior treatment. The result showed that LH levels after the cerebral administration (ad) tended to increase compared to the levels before ad. In MBH, LH levels in 4 cases (4/5), rose and were not changed in 1 case at 0.5 - 2 hours after ad compared to those before ad. There were no significant changes at 2.5 hours after ad. When it was injected in VM, LH levels in 3 cases (3/4) rose, and were not changed in 1 case after ad compared to those before ad. In the control, there were no changes in plasma LH levels between the pre-and post-treatment except 1 case in MBH. This study suggested that DA could up-regulate LH secretion through hypothalamus level in the castrated boars.
文摘Silencing of gene expression by RNA interference (RNAi) has become a widely used tool. For the study of mammalian gene function expression vectors for short hairpin RNA (shRNA) were developed. However the standard methods of shRNA transgenic (Tg) mice production have not been established. Sry (sex-determining region on the Y chromosome) is a mammalian sex-determining gene on the Y chromosome. In mice, the transient expression of Sry in supporting cell precursor cells between 10.5 and 12.5 days post-coitus (dpc) triggers the differentiation of Sertoli cells from granulosa cells. Then high efficiency of Sry gene silencing in Tg mice should induce XY male-to-female sex reversal. An shRNA Tg mouse targeting Sry gene was attempted to be generated by pronuclear microinjection. A low rate (Tg pups/all pups born after microinjection = 2/154 to 7/178) of Tg pups was observed. These Tg mice showed no XY male-to-female sex reversal. The results suggest that exogenous expression of small RNA might exert a negative effect on embryonic development and another approach should be needed for RNAi transgenesis in mice.
文摘Anti-keratin monoclonal antibody AF5 was introduced into fertilized eggs of Xenopus laevis., and its effects on embryonic development were studied. Survival rate of the antikeratin-injected embryos was much lower (only 35.76% at gastrula) than that of the control (74.85% at gastrula), in which embryos were injected with mouse IgG. Most of survivors in the experimental series showed aberrant external appearance. On the other hand, in cleavage stage, ie 2-7 h after fertilization, immunohistochemi-cal staining of embryos showed that the experimental embryos were mostly keratin negative, while embryos of the control ones were keratin positive. When introducing this antikeratin into one cell of a 2-cell embryo, only the unin-jected half of the embryo continued its development while the other half could not develop at all. These results suggested that intact keratin cytoskeleton in early embryos is indispensable to the embryonic development of Xenopus laevis.
基金the National Natural Sci-ence Foundation of China(Nos.31672636,31772834,and 31972774)the National Key R&D Program of China(Nos.2018YFD0901202 and 2018YFD0900202)the Key Research and Development Program of Shandong Pro-vince,China(No.2019GHY1120070)。
文摘The whole-genome sequence of the olive flounder(Paralichthys olivaceus)provides a basis for gene functional analyses,which is important for the aquaculture industry.Understanding gene function will help us to select better economic traits such as fast growth and better culture conditions,which further will increase the aquaculture output.Gene knockout is an important reverse genetics approach for in vivo studies of gene function.In this study,the CRISPR/Cas9 genome editing method with a microinjection system using a simple braked needle was employed in olive flounder.After injection in embryos,green fluorescent protein expression was detected in 40%of larvae.The proportion of normal-hatched larvae was approximately 50%.Different mutations,including short indels and fragment deletions,were found in our test genes gsdf and myomaker.Additionally,we detected more than one mutation in a single larva.In summary,our microinjection technique and CRISPR/Cas9 can be applied to study gene functions in olive flounder.
文摘Objective To create transgenic rabbits by microinjecting human apolipoprotein A-Ⅱ (apoA-Ⅱ) gene into one-cell embryos, to study apoA-Ⅱ gene function on plasma lipoprotein metabolism and atherosclerosis. Methods Superovulation and synchronization of estrus were induced in female Japanese White Rabbits by injecting hormone, then mating with male. After collected the fertilized eggs, the human apoA-Ⅱ gene was microinjected into the male pronucleus of eggs. The injected eggs were transferred into recipient female rabbits. Last, extract DNA from the new borns ear and determine whether the newborns were transgenic by polymerase chain reaction (PCR) or Southern blot analysis. Results A total of 822 embryos with microinjection of human apoAⅡ gene were implanted into 28 recipient rabbits. The number of surviving newborns was 37. 3 transgenic positive surviving founders were found with human apoA-Ⅱ.
