Highlights●CRISPR/Cas9 RNP complex-based strategy demonstrates robustness and accuracy in generating gene-edited sheep.●Sheep horn development remains unaffected by partial RXFP2 knockout.●Partial RXFP2 knockout re...Highlights●CRISPR/Cas9 RNP complex-based strategy demonstrates robustness and accuracy in generating gene-edited sheep.●Sheep horn development remains unaffected by partial RXFP2 knockout.●Partial RXFP2 knockout results in unilateral cryptorchidism in sheep.展开更多
Background:C1QL3 is widely expressed in the brain and is specifically produced by a subset of excitatory neurons.However,its function is still not clear.We established C1ql3-deficient rats to investigate the role of C...Background:C1QL3 is widely expressed in the brain and is specifically produced by a subset of excitatory neurons.However,its function is still not clear.We established C1ql3-deficient rats to investigate the role of C1QL3 in the brain.Methods:C1ql3 knockout(KO)rats were generated using CRISPR/Cas9.C1ql3 KO was determined by polymerase chain reaction(PCR),DNA sequencing,and western blot-ting.Microglia morphology and cytokine expression with or without lipopolysaccha-ride(LPS)stimulus were analyzed using immunohistochemistry and real-time PCR.The brain structure changes in KO rats were examined using magnetic resonance imaging.Neuronal architecture alteration was analyzed by performing Golgi staining.Behavior was evaluated using the open field test,Morris water maze test,and Y maze test.Results:C1ql3 KO significantly increased the number of ramified microglia and decreased the number of hypertrophic microglia,whereas C1ql3 KO did not in-fluence the expression of pro-inflammatory factors and anti-inflammatory factors except IL-10.C1ql3 KO brains had more amoeboid microglia types and higher Arg-1 expression compared with the WT rats after LPS stimulation.The brain weights and HPC sizes of C1ql3 KO rats did not differ from WT rats.C1ql3 KO damaged neuronal integrity including neuron dendritic arbors and spine density.C1ql3 KO rats demonstrated an increase in spontaneous activity and an impairment in short working memory.Conclusions:C1ql3 KO not only interrupts the neuronal integrity but also affects the microglial activation,resulting in hyperactive behavior and impaired short memory in rats,which highlights the role of C1QL3 in the regulation of structure and function of both neuronal and microglial cells.展开更多
Objective To investigate the mechanism of in alleviating colonic mucosal inflammation in ten-eleven translocation(TET)protein 2 gene knockout(TET2^(-/-))mice with ulcerative colitis(UC)by regulating DNA methyltransfer...Objective To investigate the mechanism of in alleviating colonic mucosal inflammation in ten-eleven translocation(TET)protein 2 gene knockout(TET2^(-/-))mice with ulcerative colitis(UC)by regulating DNA methyltransferase(DNMT)and DNA hydroxymethylase.Methods Male specific pathogen-free(SPF)grade C57BL/6J wild-type(WT)mice(n=8)and TET2^(-/-)mice(n=20)were used to establish UC models by freely drinking 3%dextran sulfate sodium solution for 7 d.After UC model validation through histopathological examination in two mice from each type,the remaining mice were divided into four groups(n=6 in each group):WT model(WT+UC),TET2^(-/-)model(TET2^(-/-)+UC),TET2^(-/-)mild moxibustion(TET2^(-/-)+MM),and TET2^(-/-)electroacupuncture(TET2^(-/-)+EA)groups.TET2^(-/-)+MM group received mild moxibustion on Tianshu(ST25)and Qihai(CV6)for 10 min daily for 7 d.The TET2^(-/-)+EA group also applied electroacupuncture(1 mA,2/100 Hz)at the same acupoints for 10 min daily for 7 d.The disease activity index(DAI)scores of each group of mice were accessed daily.The colon lengths of mice in groups were measured following intervention.The pathological changes in the colon tissues were observed with hematoxylin and eosin(HE)staining.The concentrations of interleukin(IL)-6,C-C motif chemokine 17(CCL17),and C-X-C motif chemokine ligand 10(CXCL10)in serum were detected by enzyme-linked immunosorbent assay(ELISA).The expression of DNMT proteins(DNMT1,DNMT3A,and DNMT3B)in the colon tissues was detected by immunohistochemistry.The expression of 5-methylcytosine(5-mC),5-hydroxymethylcytosine(5-hmC),histone deacetylase 2(HDAC2),and DNA hydroxymethylase family proteins(TET 1 and TET3)was detected using immunofluorescence,which also determined the co-localization of TET1 and IL-6 protein.Results Compared with WT+UC group,TET2^(-/-)+UC group exhibited significantly higher DAI scores and shorter colon lengths(P<0.01).Both mild moxibustion and electroacupuncture significantly decreased DAI scores and ameliorated colon shortening in TET2^(-/-)mice(P<0.001).Histopathological scores of TET2^(-/-)+UC mice were significantly higher than those of WT+UC group(P<0.001)and were significantly reduced after both mild moxibustion and electroacupuncture interventions(P<0.001).Serum levels of IL-6,CCL17,and CXCL10 were significantly elevated in TET2^(-/-)+UC group compared with WT+UC group(P<0.001).Mild moxibustion significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.001,P<0.001,and P<0.01,respectively),while electroacupuncture also significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.05,P<0.01,and P<0.01,respectively).TET2^(-/-)+UC mice showed increased expression levels of DNMT1,DNMT3A,DNMT3B,and 5-mC(P<0.05,P<0.01 and P<0.001,respectively),with decreased expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001).Mild moxibustion significantly reduced DNMT1,DNMT3B,and 5-mC levels(P<0.05,P<0.01,and P<0.001,respectively),while increasing expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001,P<0.001,P<0.05,and P<0.001,respectively).Electroacupuncture significantly decreased 5-mC and DNMT3B levels(P<0.001 and P<0.01,respectively)and increased 5-hmC and HDAC2 levels(P<0.05 and P<0.001,respectively),but did not significantly affect TET1 and TET3 expression(P>0.05).Compared with TET2^(-/-)+MM group,TET2^(-/-)+EA group showed significantly higher 5-mC expression(P<0.001).TET2^(-/-)+UC group exhibited markedly increased IL-6 expression and higher co-localization of TET1 and IL-6 in mucosal epithelium,whereas minimal IL-6 expression was observed in the other groups.Conclusion Mild moxibustion and electroacupuncture significantly ameliorate colonic inflammation exacerbated by TET2 deficiency in UC mice via epigenetic modulation.Distinct mechanisms exist between the two interventions:mild moxibustion regulates both DNMT and hydroxymethylase,whereas electroacupuncture primarily affects DNMT.展开更多
Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestat...Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.展开更多
Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and NPY8BR.Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest th...Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and NPY8BR.Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation;this has been found in only a few fish,at present.In order to better understand the physiological function of npy8br,especially in digestion,we used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to generate npy8br-/-japanese medaka(Oryzias latipes).We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability,ultimately affecting their growth.Specifically,npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes(npy and agrp).npy8br-/-medaka larvae fed for 10 d(10th day of feeding)still had incompletely digested brine shrimp(Artemia nauplii)in the digestive tract 8 h after feeding,the messenger RNA(mRNA)expression levels of digestion-related genes(amy,lpl,ctra,and ctrb)were significantly decreased,and the activity of amylase,trypsin,and lipase also significantly decreased.The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes(gh and igf1).Hematoxylin and eosin(H&E)sections of intestinal tissue showed that npy8br-/-medaka larvae had damaged intestine,thinned intestinal wall,and shortened intestinal villi.So far,this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.展开更多
AIM:To investigate the role of transmembrane protein 206(TMEM206)in corneal edema in mice by knockout the TMEM206 gene using CRISPR/Cas9 editing technology.METHODS:TMEM206-knockout mice were generated using the CRISPR...AIM:To investigate the role of transmembrane protein 206(TMEM206)in corneal edema in mice by knockout the TMEM206 gene using CRISPR/Cas9 editing technology.METHODS:TMEM206-knockout mice were generated using the CRISPR-Cas9 system.Variations in ophthalmic pathology were observed using slit lamp microscope and optical coherence tomography(OCT),intraocular pressure(IOP)was measured using a TonoLab Rebound Tonometer,and the ultrastructure of the corneal was observed using a transmission electron microscope.RESULTS:Corneal opacity was observed in 4/18 homozygous TMEM206^(-/-)mice whereas a similar change was not observed in heterozygous TMEM206^(+/-)mice and wild-type littermates.OCT examination showed that the mean central cornea thickness was 125±5.4μm in 4 homozygous TMEM206^(-/-)mice developed corneal edema and 115±1.2μm in wild-type mice(t=3.468,P<0.05)at 43wk.The mean IOP was 12.08±0.07 mm Hg in four right eyes with corneal edema and 12.03±0.03 mm Hg in three normal left eyes(P>0.05).Transmission electron microscopy revealed a disruption in the organization of the collagen fibrils in the central part of the cornea in homozygous TMEM206^(-/-)mice.CONCLUSION:TMEM206 is associated with corneal edema which caused organizational disruption of collagen fibrils in corneas of mice.展开更多
Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host...Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host’s immune system is compromised.Rhoptry proteins are major virulence factors that enable T.gondii to invade host cells.This study aims to construct a T.gondii rhoptry protein 41(rop41/ROP41)gene knockout strain and preliminarily investigate the biological function of rop41.Methods:Using CRISPR/Cas9 technology,a specific single-guide RNA(sgRNA)for the target gene was designed and linked to a recombinant plasmid.Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes.The recombinant plasmid and the homologous fragments were electroporated into T.gondii,and PCR identification was performed after drug selection and monoclonal screening.Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T.gondii in host cells.Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity.The STRING database was utilized to construct a protein-protein interaction(PPI)network,and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.Results:The T.gondii rop41 gene knockout strain(RHΔku80Δrop41)was successfully constructed and stably inherited.Plaque assays showed that compared with the parental strain,the number of plaques formed by the rop41 gene knockout strain did not significantly decrease,but the reduction in plaque size was statistically significant(P<0.05).After the rop41 gene was knocked out,the invasion ability of T.gondii was reduced,but there was no statistically significant difference in its proliferation ability(P>0.05).The PPI network revealed that ROP41 was associated with other protein kinases and autophagy related proteins.Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction,biosynthesis,metabolism,and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.Conclusion:The T.gondii RHΔku80Δrop41 strain has been successfully constructed.ROP41 primarily affects the ability of T.gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T.gondii and the host.展开更多
[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification...[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.展开更多
Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this ...Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation,as well as expanding the potential pool of usable donor grafts.The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes,increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis.Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury.IRI involves a complex interplay between neutrophils,natural killer T-cells cells,CD4+ T cell subtypes,cytokines,nitric oxide synthases,haem oxygenase-1,survival kinases such as the signal transducer and activator of transcription,Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways.Transgenic animals,particularly genetic knockout models,have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies.Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein.This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.展开更多
The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their ...The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon （apyrF） as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding apyrF host. After positive selection of the single-crossover integration recombinants （pop-in） on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants （pop-out） with 5-fluoroorotic acid （5-FOA）, the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.展开更多
Insulin resistance is a hallmark of type 2 diabetes. In an effort to understand and treat this condition, re searchers have used genetic manipulation of mice to uncover insulin signaling pathways and determine the eff...Insulin resistance is a hallmark of type 2 diabetes. In an effort to understand and treat this condition, re searchers have used genetic manipulation of mice to uncover insulin signaling pathways and determine the effects of their perturbation. After decades of research much has been learned, but the pathophysiology o insulin resistance in human diabetes remains contro versial, and treating insulin resistance remains a chal lenge. This review will discuss limitations of mouse models lacking select insulin signaling molecule genes In the most influential mouse models, glucose metabo lism differs from that of humans at the cellular, organ and whole-organism levels, and these differences limi the relevance and benefit of the mouse models both in terms of mechanistic investigations and therapeutic development. These differences are due partly to im mutable differences in mouse and human biology, and partly to the failure of genetic modifications to produce an accurate model of human diabetes. Several fac tors often limit the mechanistic insights gained from experimental mice to the particular species and strain including: developmental effects, unexpected meta bolic adjustments, genetic background effects, and technical issues. We conclude that the limitations and weaknesses of genetically modified mouse models of insulin resistance underscore the need for redirection of research efforts toward methods that are more directly relevant to human physiology.展开更多
The mechanism of androgen action is complex. Recently, significant advances have been made into our understanding of how androgens act via the androgen receptor （AR） through the use of genetically modified mouse mod...The mechanism of androgen action is complex. Recently, significant advances have been made into our understanding of how androgens act via the androgen receptor （AR） through the use of genetically modified mouse models. A number of global and tissue-specific AR knockout （ARKO） models have been generated using the Cre-loxP system which allows tissue- and/or cell-specific deletion. These ARKO models have examined a number of sites of androgen action including the cardiovascular system, the immune and hemopoetic system, bone, muscle, adipose tissue, the prostate and the brain. This review focuses on the insights that have been gained into human androgen deficiency through the use of ARKO mouse models at each of these sites of action, and highlights the strengths and limitations of these Cre-loxP mouse models that should be considered to ensure accurate interpretation of the phenotype.展开更多
Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first ...Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first commercial hybrid maize was introduced in the 1930s（Duvick,2001）.展开更多
BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remai...BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.展开更多
Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult hom...Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.展开更多
基金supported by the National Key Research and Development Program of China(2022YFD1300200)the National Natural Science Foundation of China(32161143010,32202646,and 32272848)+2 种基金the China Agriculture Research System(CARS-39)the Key Special Project of Ningxia Science and Technology Department,China(2021BEF02024)the local grants,China(NXTS2021-001,2022GD-TSLD-46,NK2022010207,and NXTS2022-001)。
文摘Highlights●CRISPR/Cas9 RNP complex-based strategy demonstrates robustness and accuracy in generating gene-edited sheep.●Sheep horn development remains unaffected by partial RXFP2 knockout.●Partial RXFP2 knockout results in unilateral cryptorchidism in sheep.
基金The present work was supported by the National Natural Science Foundation(31970508)the National Key Research and Development Program of China(2022YFF0710702).
文摘Background:C1QL3 is widely expressed in the brain and is specifically produced by a subset of excitatory neurons.However,its function is still not clear.We established C1ql3-deficient rats to investigate the role of C1QL3 in the brain.Methods:C1ql3 knockout(KO)rats were generated using CRISPR/Cas9.C1ql3 KO was determined by polymerase chain reaction(PCR),DNA sequencing,and western blot-ting.Microglia morphology and cytokine expression with or without lipopolysaccha-ride(LPS)stimulus were analyzed using immunohistochemistry and real-time PCR.The brain structure changes in KO rats were examined using magnetic resonance imaging.Neuronal architecture alteration was analyzed by performing Golgi staining.Behavior was evaluated using the open field test,Morris water maze test,and Y maze test.Results:C1ql3 KO significantly increased the number of ramified microglia and decreased the number of hypertrophic microglia,whereas C1ql3 KO did not in-fluence the expression of pro-inflammatory factors and anti-inflammatory factors except IL-10.C1ql3 KO brains had more amoeboid microglia types and higher Arg-1 expression compared with the WT rats after LPS stimulation.The brain weights and HPC sizes of C1ql3 KO rats did not differ from WT rats.C1ql3 KO damaged neuronal integrity including neuron dendritic arbors and spine density.C1ql3 KO rats demonstrated an increase in spontaneous activity and an impairment in short working memory.Conclusions:C1ql3 KO not only interrupts the neuronal integrity but also affects the microglial activation,resulting in hyperactive behavior and impaired short memory in rats,which highlights the role of C1QL3 in the regulation of structure and function of both neuronal and microglial cells.
基金National Natural Science Foundation of China(82274641,81873372,and 82105012).
文摘Objective To investigate the mechanism of in alleviating colonic mucosal inflammation in ten-eleven translocation(TET)protein 2 gene knockout(TET2^(-/-))mice with ulcerative colitis(UC)by regulating DNA methyltransferase(DNMT)and DNA hydroxymethylase.Methods Male specific pathogen-free(SPF)grade C57BL/6J wild-type(WT)mice(n=8)and TET2^(-/-)mice(n=20)were used to establish UC models by freely drinking 3%dextran sulfate sodium solution for 7 d.After UC model validation through histopathological examination in two mice from each type,the remaining mice were divided into four groups(n=6 in each group):WT model(WT+UC),TET2^(-/-)model(TET2^(-/-)+UC),TET2^(-/-)mild moxibustion(TET2^(-/-)+MM),and TET2^(-/-)electroacupuncture(TET2^(-/-)+EA)groups.TET2^(-/-)+MM group received mild moxibustion on Tianshu(ST25)and Qihai(CV6)for 10 min daily for 7 d.The TET2^(-/-)+EA group also applied electroacupuncture(1 mA,2/100 Hz)at the same acupoints for 10 min daily for 7 d.The disease activity index(DAI)scores of each group of mice were accessed daily.The colon lengths of mice in groups were measured following intervention.The pathological changes in the colon tissues were observed with hematoxylin and eosin(HE)staining.The concentrations of interleukin(IL)-6,C-C motif chemokine 17(CCL17),and C-X-C motif chemokine ligand 10(CXCL10)in serum were detected by enzyme-linked immunosorbent assay(ELISA).The expression of DNMT proteins(DNMT1,DNMT3A,and DNMT3B)in the colon tissues was detected by immunohistochemistry.The expression of 5-methylcytosine(5-mC),5-hydroxymethylcytosine(5-hmC),histone deacetylase 2(HDAC2),and DNA hydroxymethylase family proteins(TET 1 and TET3)was detected using immunofluorescence,which also determined the co-localization of TET1 and IL-6 protein.Results Compared with WT+UC group,TET2^(-/-)+UC group exhibited significantly higher DAI scores and shorter colon lengths(P<0.01).Both mild moxibustion and electroacupuncture significantly decreased DAI scores and ameliorated colon shortening in TET2^(-/-)mice(P<0.001).Histopathological scores of TET2^(-/-)+UC mice were significantly higher than those of WT+UC group(P<0.001)and were significantly reduced after both mild moxibustion and electroacupuncture interventions(P<0.001).Serum levels of IL-6,CCL17,and CXCL10 were significantly elevated in TET2^(-/-)+UC group compared with WT+UC group(P<0.001).Mild moxibustion significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.001,P<0.001,and P<0.01,respectively),while electroacupuncture also significantly reduced IL-6,CCL17,and CXCL10 levels(P<0.05,P<0.01,and P<0.01,respectively).TET2^(-/-)+UC mice showed increased expression levels of DNMT1,DNMT3A,DNMT3B,and 5-mC(P<0.05,P<0.01 and P<0.001,respectively),with decreased expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001).Mild moxibustion significantly reduced DNMT1,DNMT3B,and 5-mC levels(P<0.05,P<0.01,and P<0.001,respectively),while increasing expression levels of TET1,TET3,5-hmC,and HDAC2(P<0.001,P<0.001,P<0.05,and P<0.001,respectively).Electroacupuncture significantly decreased 5-mC and DNMT3B levels(P<0.001 and P<0.01,respectively)and increased 5-hmC and HDAC2 levels(P<0.05 and P<0.001,respectively),but did not significantly affect TET1 and TET3 expression(P>0.05).Compared with TET2^(-/-)+MM group,TET2^(-/-)+EA group showed significantly higher 5-mC expression(P<0.001).TET2^(-/-)+UC group exhibited markedly increased IL-6 expression and higher co-localization of TET1 and IL-6 in mucosal epithelium,whereas minimal IL-6 expression was observed in the other groups.Conclusion Mild moxibustion and electroacupuncture significantly ameliorate colonic inflammation exacerbated by TET2 deficiency in UC mice via epigenetic modulation.Distinct mechanisms exist between the two interventions:mild moxibustion regulates both DNMT and hydroxymethylase,whereas electroacupuncture primarily affects DNMT.
基金financially supported by the National Research Foundation of Korea(NRF-2021R1A5A1033157 for SRC program:382 Comparative medicine Disease Research Center,NRF-2021R1F1A105195313)the Research Institute of Veterinary Science,the BK21 Four for Future Veterinary Medicine Leading Education and Research Center,and a Seoul National University(SNU)grant(#550e2020005)。
文摘Background Genome editing has been considered as powerful tool in agricultural fields.However,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising cost.Furthermore,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in cattle.In this point of view,electroporation in embryos has been risen as an alternative.Results First,editing efficiency of our electroporation methods were tested for embryos.Presence of mutation on embryo was confirmed by T7E1 assay.With first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,respectively.In case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for BLG.Afterwards,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep sequencing.Thirteen recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double KO.Ten surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both genes.Conclusions These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively applied.Finally,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.
基金supported by the National Natural Science Foundation of China(No.31972809)the Key Research&Development Program of Hubei Province(No.2022BBA0051),China.
文摘Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and NPY8BR.Changes in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation;this has been found in only a few fish,at present.In order to better understand the physiological function of npy8br,especially in digestion,we used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to generate npy8br-/-japanese medaka(Oryzias latipes).We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability,ultimately affecting their growth.Specifically,npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes(npy and agrp).npy8br-/-medaka larvae fed for 10 d(10th day of feeding)still had incompletely digested brine shrimp(Artemia nauplii)in the digestive tract 8 h after feeding,the messenger RNA(mRNA)expression levels of digestion-related genes(amy,lpl,ctra,and ctrb)were significantly decreased,and the activity of amylase,trypsin,and lipase also significantly decreased.The deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes(gh and igf1).Hematoxylin and eosin(H&E)sections of intestinal tissue showed that npy8br-/-medaka larvae had damaged intestine,thinned intestinal wall,and shortened intestinal villi.So far,this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.
基金Supported by National Natural Science Foundation of China(No.31571294).
文摘AIM:To investigate the role of transmembrane protein 206(TMEM206)in corneal edema in mice by knockout the TMEM206 gene using CRISPR/Cas9 editing technology.METHODS:TMEM206-knockout mice were generated using the CRISPR-Cas9 system.Variations in ophthalmic pathology were observed using slit lamp microscope and optical coherence tomography(OCT),intraocular pressure(IOP)was measured using a TonoLab Rebound Tonometer,and the ultrastructure of the corneal was observed using a transmission electron microscope.RESULTS:Corneal opacity was observed in 4/18 homozygous TMEM206^(-/-)mice whereas a similar change was not observed in heterozygous TMEM206^(+/-)mice and wild-type littermates.OCT examination showed that the mean central cornea thickness was 125±5.4μm in 4 homozygous TMEM206^(-/-)mice developed corneal edema and 115±1.2μm in wild-type mice(t=3.468,P<0.05)at 43wk.The mean IOP was 12.08±0.07 mm Hg in four right eyes with corneal edema and 12.03±0.03 mm Hg in three normal left eyes(P>0.05).Transmission electron microscopy revealed a disruption in the organization of the collagen fibrils in the central part of the cornea in homozygous TMEM206^(-/-)mice.CONCLUSION:TMEM206 is associated with corneal edema which caused organizational disruption of collagen fibrils in corneas of mice.
基金supported by the National Natural Science Foundation of China(32170510)the Innovation Training Program of Central South University(20240026020055),China.
文摘Objective:Toxoplasmosis is a zoonotic parasitic disease caused by Toxoplasma gondii(T.gondii),which can lead to complications such as encephalitis and ocular toxoplasmosis.The disease becomes more severe when the host’s immune system is compromised.Rhoptry proteins are major virulence factors that enable T.gondii to invade host cells.This study aims to construct a T.gondii rhoptry protein 41(rop41/ROP41)gene knockout strain and preliminarily investigate the biological function of rop41.Methods:Using CRISPR/Cas9 technology,a specific single-guide RNA(sgRNA)for the target gene was designed and linked to a recombinant plasmid.Homologous fragments were fused with a pyrimethamine resistance gene for selection purposes.The recombinant plasmid and the homologous fragments were electroporated into T.gondii,and PCR identification was performed after drug selection and monoclonal screening.Plaque assays were used to comprehensively assess whether rop41 affected the growth and proliferation of T.gondii in host cells.Invasion and proliferation assays were conducted to evaluate the invasion ability of the knockout strain into host cells and its intracellular proliferation capacity.The STRING database was utilized to construct a protein-protein interaction(PPI)network,and functional enrichment analysis was performed to predict the signaling pathways in which ROP41 might be involved.Results:The T.gondii rop41 gene knockout strain(RHΔku80Δrop41)was successfully constructed and stably inherited.Plaque assays showed that compared with the parental strain,the number of plaques formed by the rop41 gene knockout strain did not significantly decrease,but the reduction in plaque size was statistically significant(P<0.05).After the rop41 gene was knocked out,the invasion ability of T.gondii was reduced,but there was no statistically significant difference in its proliferation ability(P>0.05).The PPI network revealed that ROP41 was associated with other protein kinases and autophagy related proteins.Enrichment analysis indicated that proteins interacting with ROP41 may be involved in signal transduction,biosynthesis,metabolism,and autophagy-related pathways and could be components of various kinase complexes and phagocytic vesicles.Conclusion:The T.gondii RHΔku80Δrop41 strain has been successfully constructed.ROP41 primarily affects the ability of T.gondii to invade host cells and may play a role in signal transduction and autophagy-related pathways between T.gondii and the host.
基金Supported by the National Natural Science Fundation Item of China(30970578,31070651)"Excellent Talent Support Plan in NewCentury"of Ministry of Education(NECT-08-0731)~~
文摘[Objective] The research aimed to construct the prokaryotic expression vector of VP5 protein of IBDV.The transmembrane region sequence of VP5 protein was knocked out.Moreover,the expression,separation and purification of objective protein were carried out.[Method] PCR technology was used to respectively amplify the extracellular and intracellular fragments of VP5 gene of IBDV.Then,the two fragments were simultaneously linked to pET-28b（＋）,and it was the vector-intracellular fragment-extracellular fragment-vector.The recombinant expression plasmid pET-VP5-FC and the improved pET-VP5-SC of VP5 whose transmembrane region gene fragment was knocked out were constructed.Then,the expression plasmid was transformed into BL21（DE3）.After IPTG induction,the recombinant protein was purified by Ni affinity chromatography and the gel filtration chromatography.[Result] The soluble expressed VP5 of IBDV was obtained.[Conclusion] The research laid the foundation for further studying the structure and function of VP5 protein.
文摘Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation surgery.Understanding the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation,as well as expanding the potential pool of usable donor grafts.The initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes,increased apoptosis and acute inflammatory changes with increased hepatocyte necrosis.Some adaptive pathways are activated during reperfusion that reduce the reperfusion injury.IRI involves a complex interplay between neutrophils,natural killer T-cells cells,CD4+ T cell subtypes,cytokines,nitric oxide synthases,haem oxygenase-1,survival kinases such as the signal transducer and activator of transcription,Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ pathways.Transgenic animals,particularly genetic knockout models,have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological studies.Targeted disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same protein.This article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.
基金supported by the National Natural Science Foundation of China(Nos.30830004,30925001)the National 863 Program of China(No.2009AA09Z401)the Chinese Academy of Sciences(No.KSCX2-EW-G-2)
文摘The haloarchaea Haloferax mediterranei and Haloarcula hispanica are both polyhydroxyalkanoate producers in the domain Archaea, and they are becoming increasingly attractive for research and biotechnology due to their unique genetic and metabolic features. To accelerate their genome-level genetic and metabolic analyses, we have developed specific and highly efficient gene knockout systems for these two haloarchaea. These gene knockout systems consist of a suicide plasmid vector with the pyrF gene as the selection marker and a uracil auxotrophic haloarchaeon （apyrF） as the host. For in-frame deletion of a target gene, the suicide plasmid carrying the flanking region of the target gene was transferred into the corresponding apyrF host. After positive selection of the single-crossover integration recombinants （pop-in） on AS-168SY medium without uracil and counterselection of the double-crossover pyrF-excised recombinants （pop-out） with 5-fluoroorotic acid （5-FOA）, the target gene knockout mutants were confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of these systems by knocking out the crtB gene which encodes a phytoene synthase in these haloarchaea. In conclusion, these well-developed knockout systems would greatly accelerate the functional genomic research of these halophilic archaea.
文摘Insulin resistance is a hallmark of type 2 diabetes. In an effort to understand and treat this condition, re searchers have used genetic manipulation of mice to uncover insulin signaling pathways and determine the effects of their perturbation. After decades of research much has been learned, but the pathophysiology o insulin resistance in human diabetes remains contro versial, and treating insulin resistance remains a chal lenge. This review will discuss limitations of mouse models lacking select insulin signaling molecule genes In the most influential mouse models, glucose metabo lism differs from that of humans at the cellular, organ and whole-organism levels, and these differences limi the relevance and benefit of the mouse models both in terms of mechanistic investigations and therapeutic development. These differences are due partly to im mutable differences in mouse and human biology, and partly to the failure of genetic modifications to produce an accurate model of human diabetes. Several fac tors often limit the mechanistic insights gained from experimental mice to the particular species and strain including: developmental effects, unexpected meta bolic adjustments, genetic background effects, and technical issues. We conclude that the limitations and weaknesses of genetically modified mouse models of insulin resistance underscore the need for redirection of research efforts toward methods that are more directly relevant to human physiology.
文摘The mechanism of androgen action is complex. Recently, significant advances have been made into our understanding of how androgens act via the androgen receptor （AR） through the use of genetically modified mouse models. A number of global and tissue-specific AR knockout （ARKO） models have been generated using the Cre-loxP system which allows tissue- and/or cell-specific deletion. These ARKO models have examined a number of sites of androgen action including the cardiovascular system, the immune and hemopoetic system, bone, muscle, adipose tissue, the prostate and the brain. This review focuses on the insights that have been gained into human androgen deficiency through the use of ARKO mouse models at each of these sites of action, and highlights the strengths and limitations of these Cre-loxP mouse models that should be considered to ensure accurate interpretation of the phenotype.
基金supported by grants from the National Natural Science Foundation of China (Nos. 31501376 and 31570369)the National Key Research and Development Program of China (No. 2016YFD0101804)the National Transgenic Science and Technology Program (No. 2016ZX08010002)
文摘Maize（Zea mays L.）is one of the most important cereal crops,with a global production of 1.02 billion tons in 2013（Baldaufa et al.,2016）.Heterosis is widely used to increase the productivity of maize,and the first commercial hybrid maize was introduced in the 1930s（Duvick,2001）.
基金the Natural Science Foundation of Shandong Province,No. Y2008C54
文摘BACKGROUND： Previous studies have shown that p75 neurotrophin receptor plays an important role in peripheral nerve injury. However, the role of p75 neurotrophin receptor in the regeneration of peripheral nerves remains poorly understood. OBJECTIVE： To study the effect of p75 neurotrophin receptor on facial nerve regeneration. DESIGN, TIME AND SETTING： A randomized controlled experiment was performed in the Regeneration Laboratory of Flinders University, Australia and the Biomedical Laboratory of Dentistry School, Shandong University from March 2005 to February 2006. MATERIALS： Cholera toxin B subunit, fast blue, and biotin rabbit-anti goat IgG were provided by Sigma, USA; goat-anti choleratoxin B subunit ant/body was provided by List Biologicals, USA. METHODS： In p75 neurotrophin receptor knockout and wild type 129/sv mice, the facial nerves on one side were crushed. At days 2 and 4 following injury, regenerating motor neurons in the facial nuclei were labeled by fast blue, and the regenerating axon was labeled by the anterograde tracer choleratoxin B subunit. MAIN OUTCOME MEASURES： Axonal regenerative velocity and number were detected by immunohistochemical staining of choleratoxin B subunit, growth-associated protein, protein gene product 9.5, and calcitonin-gene-related peptide; survival of motor neurons in the facial nuclei was detected by retrograde fast blue. RESULTS： Axonal growth in the facial nerve of p75 neurotrophin receptor knockout mice was significantly less than in wild type mice. At day 7 after injury, the number of regenerating motor neurons in p75 neurotrophin receptor knockout mice remained significantly less than in wild type mice （P 〈 0.05）. The number of positively stained fibers for growth-associated protein-43, protein gene product 9.5, and calcitonin-gene-related peptide in p75 neurotrophin receptor knockout mice was significantly less than in wild type mice （P 〈 0.01）. CONCLUSION： p75 neurotrophin receptor promoted axonal regeneration and enhanced the survival rate of motor neurons following facial nerve injury.
文摘Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.
