Phosphatase of regenerating liver-3 (PRL-3) is a member of the protein tyrosine phosphatase (PTP) superfamily and is highly expressed in cancer metastases. For better understanding of the role of PRL-3 in tumor me...Phosphatase of regenerating liver-3 (PRL-3) is a member of the protein tyrosine phosphatase (PTP) superfamily and is highly expressed in cancer metastases. For better understanding of the role of PRL-3 in tumor metastasis, we applied a rapid and efficient method for generating PRL-3 floxed mice and investigated its phenotypes. A BAC retrieval strategy was applied to construct the PRL-3 conditional gene-targeting vector. Exon 4 was selected for deletion to generate a nonfunctional prematurely terminated short peptide as it will cause a frame-shift mutation. Conditional knockout PRL-3 mice were generated by using the Cre-loxP system and were validated by Southern blot and RT-PCR analysis. Further analysis revealed the phenotype characteristics of PRL-3 knockout mice and wildtype mice. In this study, we successfully constructed the PRL-3 conditional knockout mice, which will be helpful to clarify the roles of PRL-3 and the mechanisms in tumor metastasis.展开更多
Dendrites play irreplaceable roles in the nerve conduction pathway and are vulnerable to various insults.Peripheral axotomy of motor neurons results in the retraction of dendritic arbors,and the dendritic arbor can be...Dendrites play irreplaceable roles in the nerve conduction pathway and are vulnerable to various insults.Peripheral axotomy of motor neurons results in the retraction of dendritic arbors,and the dendritic arbor can be re-expanded when reinnervation is allowed.RhoA is a target that regulates the cytoskeleton and promotes neuronal survival and axon regeneration.However,the role of RhoA in dendrite degeneration and regeneration is unknown.In this study,we explored the potential role of RhoA in dendrites.A line of motor neuronal conditional knockout mice was developed by crossbreeding HB9~(Cre+)mice with RhoA~(flox/flox)mice.We established two models for assaying dendrite degeneration and regeneration,in which the brachial plexus was transection or crush injured,respectively.We found that at 28 days after brachial plexus transection,the density,complexity,and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice were slightly decreased compared with that in Cre mice.Dendrites underwent degeneration at 7 and 14 days after brachial plexus transection and recovered at 28–56 days.The density,complexity,and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice recovered compared with results in Cre mice.These findings suggest that RhoA knockout in motor neurons attenuates dendrite degeneration and promotes dendrite regeneration after peripheral nerve injury.展开更多
Background and aim:Glucocorticoids are the only first-line drugs for severe alcoholic hepatitis(AH),with limited efficacy and various side effects on extrahepatic tissues.Liver-targeting glucocorticoid therapy may hav...Background and aim:Glucocorticoids are the only first-line drugs for severe alcoholic hepatitis(AH),with limited efficacy and various side effects on extrahepatic tissues.Liver-targeting glucocorticoid therapy may have multiple advantages over systemic glucocorticoid for AH.The aim of this study was to determine the role of hepatocellular glucocorticoid receptor(GR)in alcohol-associated steatosis(AS)and AH.Materials and methods:AS was induced by a high-fat diet plus binge alcohol in adult male and female mice with liver-specific knockout(LKO)and heterozygote of GR.AH was induced by chronic-plus-binge in middle-aged male mice with liver-specific knockin of GR.Changes in hepatic mRNA and protein expression were determined by quantitative real-time polymerase chain reaction and Western blot.Results:GR-LKO aggravated steatosis and decreased hepatic expression and circulating levels of albumin in both genders of AS mice but only increased markers of liver injury in male AS mice.Marked steatosis in GR-LKO mice was associated with induction of lipogenic genes and down-regulation of bile acid synthetic genes.Hepatic protein levels of GR,hepatocyte nuclear factor 4 alpha,and phosphorylated signal transducer and activator of transcription 3 were gene-dosage-dependently decreased,whereas that of lipogenic ATP citrate lyase was increased in male GR heterozygote and LKO mice.Interestingly,hepatic expression of estrogen receptor alpha(ERα)was induced,and the essential estrogen-inactivating enzyme sulfotransferase 1e1 was gene-dosage-dependently down-regulated in GR heterozygote and knockout AS mice,which was associated with induction of ERα-target genes.Liver-specific knockin of GR protected against liver injury and steatohepatitis in middle-aged AH mice.Conclusions:Hepatic GR deficiency plays a crucial role in the pathogenesis of AS induced by high-fat diet plus binge,and liver-specific overexpression/activation of GR protects against chronic-plus-binge-induced AH in middle-aged mice.Hepatocellular GR is important for protection against AS and AH.展开更多
The zebrafish has become a popular vertebrate animal model in biomedical research.However,it is still challenging to make conditional gene knockout(CKO)models in zebrafish due to the low efficiency of homologous recom...The zebrafish has become a popular vertebrate animal model in biomedical research.However,it is still challenging to make conditional gene knockout(CKO)models in zebrafish due to the low efficiency of homologous recombination(HR).Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin(KI)switch(zCKOIS)coupled with dual-color fluorescent reporters.Using this strategy,we generated hey2^zCKOIS which served as a hey2 KI reporter with EGFP expression.Upon Cre induction in targeted cells,the hey2^zCKOIS was switched to a non-functional CKO allele hey2^zCKOIS-inv associated with Tag RFP expression,enabling visualization of the CKO alleles.Thus,simplification of the design,and the visibility and combination of both CKO and KI alleles make our z CKOIS strategy an applicable CKO approach for zebrafish.展开更多
Vision depends on accurate signal conduction from the retina to the brain through the optic nerve,an important part of the central nervous system that consists of bundles of axons originating from retinal ganglion cel...Vision depends on accurate signal conduction from the retina to the brain through the optic nerve,an important part of the central nervous system that consists of bundles of axons originating from retinal ganglion cells.The mammalian optic nerve,an important part of the central nervous system,cannot regenerate once it is injured,leading to permanent vision loss.To date,there is no clinical treatment that can regenerate the optic nerve and restore vision.Our previous study found that the mobile zinc(Zn^(2+))level increased rapidly after optic nerve injury in the retina,specifically in the vesicles of the inner plexiform layer.Furthermore,chelating Zn^(2+)significantly promoted axonal regeneration with a long-term effect.In this study,we conditionally knocked out zinc transporter 3(ZnT3)in amacrine cells or retinal ganglion cells to construct two transgenic mouse lines(VGAT^(Cre)ZnT3^(fl/fl)and VGLUT2^(Cre)ZnT3^(fl/fl),respectively).We obtained direct evidence that the rapidly increased mobile Zn^(2+)in response to injury was from amacrine cells.We also found that selective deletion of ZnT3 in amacrine cells promoted retinal ganglion cell survival and axonal regeneration after optic nerve crush injury,improved retinal ganglion cell function,and promoted vision recovery.Sequencing analysis of reginal ganglion cells revealed that inhibiting the release of presynaptic Zn^(2+)affected the transcription of key genes related to the survival of retinal ganglion cells in postsynaptic neurons,regulated the synaptic connection between amacrine cells and retinal ganglion cells,and affected the fate of retinal ganglion cells.These results suggest that amacrine cells release Zn^(2+)to trigger transcriptomic changes related to neuronal growth and survival in reginal ganglion cells,thereby influencing the synaptic plasticity of retinal networks.These results make the theory of zinc-dependent retinal ganglion cell death more accurate and complete and provide new insights into the complex interactions between retinal cell networks.展开更多
基金supported by Grants from MOST of China (No. 2005CB522501, No.2006BAI23B00, and No.2006CB943503) to Xiang Gao
文摘Phosphatase of regenerating liver-3 (PRL-3) is a member of the protein tyrosine phosphatase (PTP) superfamily and is highly expressed in cancer metastases. For better understanding of the role of PRL-3 in tumor metastasis, we applied a rapid and efficient method for generating PRL-3 floxed mice and investigated its phenotypes. A BAC retrieval strategy was applied to construct the PRL-3 conditional gene-targeting vector. Exon 4 was selected for deletion to generate a nonfunctional prematurely terminated short peptide as it will cause a frame-shift mutation. Conditional knockout PRL-3 mice were generated by using the Cre-loxP system and were validated by Southern blot and RT-PCR analysis. Further analysis revealed the phenotype characteristics of PRL-3 knockout mice and wildtype mice. In this study, we successfully constructed the PRL-3 conditional knockout mice, which will be helpful to clarify the roles of PRL-3 and the mechanisms in tumor metastasis.
基金the Ministry of Science and Technology China Brain Initiative Grant,No.2022ZD0204701the National Natural Science Foundation of China,Nos.82071386&81870982(all to JG)。
文摘Dendrites play irreplaceable roles in the nerve conduction pathway and are vulnerable to various insults.Peripheral axotomy of motor neurons results in the retraction of dendritic arbors,and the dendritic arbor can be re-expanded when reinnervation is allowed.RhoA is a target that regulates the cytoskeleton and promotes neuronal survival and axon regeneration.However,the role of RhoA in dendrite degeneration and regeneration is unknown.In this study,we explored the potential role of RhoA in dendrites.A line of motor neuronal conditional knockout mice was developed by crossbreeding HB9~(Cre+)mice with RhoA~(flox/flox)mice.We established two models for assaying dendrite degeneration and regeneration,in which the brachial plexus was transection or crush injured,respectively.We found that at 28 days after brachial plexus transection,the density,complexity,and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice were slightly decreased compared with that in Cre mice.Dendrites underwent degeneration at 7 and 14 days after brachial plexus transection and recovered at 28–56 days.The density,complexity,and structural integrity of dendrites in the ventral horn of the spinal cord of RhoA conditional knockout mice recovered compared with results in Cre mice.These findings suggest that RhoA knockout in motor neurons attenuates dendrite degeneration and promotes dendrite regeneration after peripheral nerve injury.
基金supported by the National Institutes of Health(grant R21AA027349).
文摘Background and aim:Glucocorticoids are the only first-line drugs for severe alcoholic hepatitis(AH),with limited efficacy and various side effects on extrahepatic tissues.Liver-targeting glucocorticoid therapy may have multiple advantages over systemic glucocorticoid for AH.The aim of this study was to determine the role of hepatocellular glucocorticoid receptor(GR)in alcohol-associated steatosis(AS)and AH.Materials and methods:AS was induced by a high-fat diet plus binge alcohol in adult male and female mice with liver-specific knockout(LKO)and heterozygote of GR.AH was induced by chronic-plus-binge in middle-aged male mice with liver-specific knockin of GR.Changes in hepatic mRNA and protein expression were determined by quantitative real-time polymerase chain reaction and Western blot.Results:GR-LKO aggravated steatosis and decreased hepatic expression and circulating levels of albumin in both genders of AS mice but only increased markers of liver injury in male AS mice.Marked steatosis in GR-LKO mice was associated with induction of lipogenic genes and down-regulation of bile acid synthetic genes.Hepatic protein levels of GR,hepatocyte nuclear factor 4 alpha,and phosphorylated signal transducer and activator of transcription 3 were gene-dosage-dependently decreased,whereas that of lipogenic ATP citrate lyase was increased in male GR heterozygote and LKO mice.Interestingly,hepatic expression of estrogen receptor alpha(ERα)was induced,and the essential estrogen-inactivating enzyme sulfotransferase 1e1 was gene-dosage-dependently down-regulated in GR heterozygote and knockout AS mice,which was associated with induction of ERα-target genes.Liver-specific knockin of GR protected against liver injury and steatohepatitis in middle-aged AH mice.Conclusions:Hepatic GR deficiency plays a crucial role in the pathogenesis of AS induced by high-fat diet plus binge,and liver-specific overexpression/activation of GR protects against chronic-plus-binge-induced AH in middle-aged mice.Hepatocellular GR is important for protection against AS and AH.
基金supported by the Young Scientists Fund of the National Natural Science Foundation of China(31500849)Shanghai Municipal Science and Technology Major Project(18JC1410100,2018SHZDZX05)+3 种基金the Key Research Program of Frontier Sciences(QYZDY-SSW-SMC028)the Strategic Priority Research Program(XDB32010200)of Chinese Academy of Sciencesthe International Partnership Program,Bureau of International Co-operation of Chinese Academy of Sciences(153D31KYSB20170059)China Wan-Ren Program,and Shanghai Leading Scientist Program
文摘The zebrafish has become a popular vertebrate animal model in biomedical research.However,it is still challenging to make conditional gene knockout(CKO)models in zebrafish due to the low efficiency of homologous recombination(HR).Here we report an efficient non-HR-based method for generating zebrafish carrying a CKO and knockin(KI)switch(zCKOIS)coupled with dual-color fluorescent reporters.Using this strategy,we generated hey2^zCKOIS which served as a hey2 KI reporter with EGFP expression.Upon Cre induction in targeted cells,the hey2^zCKOIS was switched to a non-functional CKO allele hey2^zCKOIS-inv associated with Tag RFP expression,enabling visualization of the CKO alleles.Thus,simplification of the design,and the visibility and combination of both CKO and KI alleles make our z CKOIS strategy an applicable CKO approach for zebrafish.
基金the National Key R&D Project of China,No.2020YFA0112701(to YZ)the National Natural Science Foundation of China,Nos.82171057(to YZ),81870657(to YL)+1 种基金Science and Technology Program of Guangzhou of China,No.202206080005(to YZ)the Natural Science Foundation of Guangdong Province of China,No.2022A1515012168(to YL)。
文摘Vision depends on accurate signal conduction from the retina to the brain through the optic nerve,an important part of the central nervous system that consists of bundles of axons originating from retinal ganglion cells.The mammalian optic nerve,an important part of the central nervous system,cannot regenerate once it is injured,leading to permanent vision loss.To date,there is no clinical treatment that can regenerate the optic nerve and restore vision.Our previous study found that the mobile zinc(Zn^(2+))level increased rapidly after optic nerve injury in the retina,specifically in the vesicles of the inner plexiform layer.Furthermore,chelating Zn^(2+)significantly promoted axonal regeneration with a long-term effect.In this study,we conditionally knocked out zinc transporter 3(ZnT3)in amacrine cells or retinal ganglion cells to construct two transgenic mouse lines(VGAT^(Cre)ZnT3^(fl/fl)and VGLUT2^(Cre)ZnT3^(fl/fl),respectively).We obtained direct evidence that the rapidly increased mobile Zn^(2+)in response to injury was from amacrine cells.We also found that selective deletion of ZnT3 in amacrine cells promoted retinal ganglion cell survival and axonal regeneration after optic nerve crush injury,improved retinal ganglion cell function,and promoted vision recovery.Sequencing analysis of reginal ganglion cells revealed that inhibiting the release of presynaptic Zn^(2+)affected the transcription of key genes related to the survival of retinal ganglion cells in postsynaptic neurons,regulated the synaptic connection between amacrine cells and retinal ganglion cells,and affected the fate of retinal ganglion cells.These results suggest that amacrine cells release Zn^(2+)to trigger transcriptomic changes related to neuronal growth and survival in reginal ganglion cells,thereby influencing the synaptic plasticity of retinal networks.These results make the theory of zinc-dependent retinal ganglion cell death more accurate and complete and provide new insights into the complex interactions between retinal cell networks.