活化的肝星状细胞RBP-J可诱导性基因敲除小鼠的构建及应用

目的:利用Cre-loxp可诱导系统构建活化的肝星状细胞RBP-J可诱导性基因敲除小鼠模型,以实现肝纤维化过程中活化的肝星状细胞Notch信号通路的特异性阻断。方法:将Sm22αCre ERT2和RBP-Jflox/flox转基因小鼠杂交,得到F1小鼠,将繁殖的F1小鼠继续进行交配,得到F2小鼠,通过PCR鉴定出基因型为Sm22αCre ERT2,RBP-Jflox/flox的小鼠。通过腹腔注射四氯化碳建立肝纤维化模型,检测四氯化碳注射不同时间段后肝脏纤维化进展情况和肝星状细胞活化过程中Sm22α的表达情况。注射他莫昔芬诱导活化肝星状细胞中RBP-J基因的特异性敲除。分选肝星状细胞,q-PCR和Western Blot检测活化肝星状细胞中Notch下游靶基因Hes1、Hey1表达情况以验证敲除效果。结果:通过连续交配我们获得了Sm22αCre ERT2,RBP-Jflox/flox小鼠。四氯化碳腹腔注射4周即可诱导肝脏发生较为明显的纤维化,同时肝星状细胞活化并逐渐高表达Sm22α。给予他莫昔芬诱导Cre重组酶发挥作用后,敲除了活化的肝星状细胞中的RBP-J基因,其下游基因Hes1、Hey1表达降低70%以上,阻断了Notch信号通路。结论:我们成功构建了RBP-J可诱导性条件性基因敲除小鼠模型,实现了小鼠肝纤维化过程中活化肝星状细胞Notch信号通路的阻断,为深入研究Notch信号通路在肝纤维化中的作用和机制奠定了坚实的基础。     

S100a4-Cre×Ddr2flox/flox条件敲除小鼠的繁育及基因型鉴定

目的:利用Cre-LoxP重组酶系统构建成纤维细胞中Ddr2特异性敲除的纯合子小鼠并鉴定,为进一步研究Ddr2在肺纤维化中的作用提供基础。方法:将购买的Ddr2 loxp小鼠和成纤维细胞特异表达的S100a4-Cre小鼠分别繁殖与鉴定,然后将两种小鼠杂交与鉴定,最终得到基因型为Cre~× Ddr2~(flox/flox)的纯合子小鼠就是在成纤维细胞中Ddr2条件性敲除小鼠。结果:成功繁殖并用PCR技术准确鉴定了基因型为Cre~× Ddr2~(flox/flox)的纯合子小鼠,Western blot结果表明纯合子小鼠的肺成纤维细胞中Ddr2已缺失。结论:本研究利用Cre-LoxP系统成功构建了在成纤维细胞中Ddr2特异性敲除的纯合子小鼠,为进一步研究Ddr2在肺纤维化发展中的机制提供了研究平台。     

海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究

建立基于Cre/loxp重组酶系统调控的海马和新皮质特异性GABA_A受体γ2亚基(GABRG2)基因条件基因敲除小鼠模型,为深入研究海马区和新皮质GABRG2在癫痫发生中的功能作用提供动物模型。将引进的GABRG2~(fl/wt)转基因小鼠与海马和新皮质特异性表达Cre~+/+重组酶工具鼠分别进行繁配和鉴定,然后再将2种小鼠进行杂交并对其子代小鼠的基因型进行鉴定,其子代基因型为GABRG2~(fl/wt)Cre~+的小鼠为构建的海马区和新皮质特异性GABRG2基因条件性敲除小鼠。利用PCR技术鉴定小鼠基因型,Real-Time PCR和Western blot技术检测GABRG2基因在小鼠海马和新皮质中的mRNA水平和蛋白质水平的表达情况。PCR结果显示子代小鼠基因型符合GABRG2~(fl/wt)Cre~+;海马与新皮质特异性GABRG2基因敲除小鼠海马和新皮质中GABRG2的mRNA水平和蛋白质水平显著低于对照组;热造模过程中,实验组小鼠癫痫发作更明显。利用Cre/Loxp技术成功构建了海马与新皮质GABRG2基因敲除小鼠,可为进一步研究GABRG2在癫痫发生中的作用机制奠定基础。     

Dicer1 敲除对肝脏胆酸代谢和转运功能的影响

目的:微小RNA(microRNAs,miRNAs)在胆固醇的合成,代谢和转运中起着重要作用,而mi RNAs在胆固醇代谢物胆酸的代谢和转运中的作用尚不清楚。Dicer基因是miRNAs生成过程的关键酶。本课题使用肝脏特异的Dicer1基因敲除小鼠,考察肝脏Dicer1基因敲除对C57BL/6小鼠肝脏胆酸代谢和转运的影响。方法:使用白蛋白启动子驱动的Cre重组酶和Loxp系统(Alb-Cre/Loxp)在小鼠肝脏中特异的敲除Dicer1基因;分别收集3~12周龄的小鼠血液和肝脏组织,使用Cobas生化仪检测小鼠血液和肝脏中总胆酸含量;利用实时定量PCR的方法分析肝脏中胆汁酸代谢转运相关基因的表达。结果:实验发现,肝脏Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,弥漫性肝细胞轻微空泡化,偶见单个肝细胞坏死。检测胆酸代谢和转运相关基因的表达发现,胆酸合成相关基因的表达有轻度升高,但缺乏统计学差异;在肝脏细胞血管侧的胆酸摄取转运体中,Oatp1a1在Dicer1敲除小鼠肝脏中明显下调,Ntcp和Oatp1b2则无明显改变;而肝细胞血管侧胆酸外排转运体的表达均有显著升高,胆管侧的外排转运体中Abcb11表达有明显增加。结论:Dicer基因敲除后,胆酸在血液和肝脏中明显蓄积,肝脏和血液中胆酸总量显著增加。血液中胆酸的蓄积可能与肝脏细胞血管侧摄取转运体的低表达和血管侧外排转运体的高表达有关;而肝脏中胆酸的蓄积可能部分来自于轻度升高的胆酸合成酶,胆酸在肝细胞内运输途径的紊乱可能与肝脏和血液中胆酸总量的显著增加相关。     

海马与新皮质组织特异性GABRG2基因敲除小鼠模型的构建及其在遗传性癫痫伴热性惊厥附加症中的初步研究 *

建立基于Cre/loxp重组酶系统调控的海马和新皮质特异性GABA_A受体γ2亚基(GABRG2)基因条件基因敲除小鼠模型,为深入研究海马区和新皮质GABRG2在癫痫发生中的功能作用提供动物模型.将引进的GABRG2 ^fl/wt转基因小鼠与海马和新皮质特异性表达Cre ^+/+重组酶工具鼠分别进行繁配和鉴定,然后再将2种小鼠进行杂交并对其子代小鼠的基因型进行鉴定,其子代基因型为GABRG2 ^fl/wtCre ⁺的小鼠为构建的海马区和新皮质特异性GABRG2基因条件性敲除小鼠.利用PCR技术鉴定小鼠基因型,Real-Time PCR和Western blot技术检测GABRG2基因在小鼠海马和新皮质中的mRNA水平和蛋白质水平的表达情况.PCR结果显示子代小鼠基因型符合GABRG2 ^fl/wtCre ⁺;海马与新皮质特异性GABRG2基因敲除小鼠海马和新皮质中GABRG2的mRNA水平和蛋白质水平显著低于对照组;热造模过程中,实验组小鼠癫痫发作更明显.利用Cre/Loxp技术成功构建了海马与新皮质GABRG2基因敲除小鼠,可为进一步研究GABRG2在癫痫发生中的作用机制奠定基础.     

Jmjd3和Ezh2拮抗调节小鼠骨折愈合

目的:探讨Jmjd3和Ezh2在小鼠骨折愈合过程中的作用。方法:以软骨细胞条件性基因敲除8-10周龄小鼠为研究对象,按基因型随机分为6组,每组5只:其中实验组基因型为Jmjd3~(fl/fl)/Col2a1-Cre ~(ERT2),Ezh2~(fl/fl)/Col2a1-Cre ~(ERT2)或Jmjd~(3fl/fl)/Ezh2~(fl/fl)/Col2a1-Cre ~(ERT2);对照组基因型为Jmjd3~(fl/fl),Ezh2~(fl/fl)或Jmjd3~(fl/fl)/Ezh2~(fl/fl)。建立骨髓腔中插入固定针的稳定性胫骨骨折模型,于骨折术后3天、5天和7天腹腔注射Tamoxifen 3 mg/次/天。各组于术后3W处死,并于骨折部位取材行X线片及组织学检查。结果:通过连续的X线影像学及HE组织切片观察,骨折术后3周是判断小鼠骨折愈合情况的最佳时间点。X线片发现骨折术后3W时软骨细胞内Jmjd3被敲除小鼠的骨折线较对照组明显且骨化骨痂大小和密度均较低,HE切片显示骨化骨痂面积显著低于对照组,而软骨骨痂面积高于对照组;相反,X线片发现Ezh2被敲除小鼠的骨痂面积明显大于对照组,且密度高于对照组,HE组织切片显示Ezh2被敲除的小鼠的骨化骨痂的钙化程度更高,骨小梁更粗更密集。最后,X线片和HE切片均没有发现软骨细胞Jmjd3和Ezh2同时被敲除的小鼠与对照小鼠之间存在明显差异。结论:以软骨细胞特异基因敲除小鼠为基础,我们首次发现Jmjd3具有促进骨折愈合的作用,而Ezh2具有抑制骨折愈合的作用;并且发现Jmjd3和Ezh2对抗调节小鼠的骨折愈合过程,这些发现为骨折愈合治疗提供了新的分子实验基础。     

利用CRISPR-Cas9基因编辑技术敲除H9c2细胞Tudor-SN基因对细胞周期的阻滞及增殖的抑制

基因特异性敲除的细胞常用于生物学研究。近些年兴起的CRISPR-Cas9(clustered regularly interspaced short palindromic repeats-Cas9 nuclease)基因编辑技术越来越广泛地用于基因特异性敲除的实验中。本实验通过利用CRISPR-Cas9基因编辑技术,实现对大鼠心肌H9c2细胞Tudor-SN(tudor staphylococcal nuclease)基因的敲除,并观察其对H9c2细胞周期及增殖的影响。选择PX462质粒为载体,利用软件设计能特异性识别H9c2细胞Tudor-SN基因第2个外显子的上下游sgRNA(single-guided RNA),构建1对重组质粒。随后,将这对质粒共同转入H9c2细胞中,再挑选阳性单克隆细胞进行培养。Western印迹鉴定其敲除效果,并用敲除成功的细胞株通过流式细胞术及CCK-8(cell counting kit 8)实验进行细胞周期和增殖的检测。Western印迹结果显示,在阳性细胞中,Tudor-SN蛋白不表达,成功实现了对Tudor-SN基因的敲除。流式结果显示,Tudor-SN基因敲除(KO)细胞发生了G1期阻滞。G1期细胞占比由H9c2野生型(WT)细胞的54.28%±0.21%升高到KO细胞的61.96%±0.40%(*P0.05)。CCK-8实验结果显示,KO细胞的增殖速率减慢。生长第6天的A值由WT细胞的2.82±0.03降低到KO细胞1.85±0.19(*P0.05)。本实验成功构建了H9c2细胞Tudor-SN基因敲除细胞株,并检测到Tudor-SN基因敲除对细胞周期的阻滞及增殖的抑制,为研究Tudor-SN基因对心肌细胞功能的调控提供了便利的工具及研究的基础。     

去泛素化酶OTUB1肝脏特异性基因敲除小鼠模型的构建与表型分析

目的:建立OTUB1肝脏特异性基因敲除小鼠模型,初步分析其表型并研究OTUB1基因与肝脏代谢的关系。方法:利用Cre/Loxp系统构建条件性基因敲除小鼠模型,即将OTUB1~(fl/fl)转基因小鼠与Alb-Cre小鼠杂交,子代自交,得到OTUB1肝特异性基因敲除小鼠并进行鉴定。取同窝对照小鼠(control,NC)和肝特异型基因敲除(hepatic-specific OTUB1 knockout,HCKO)小鼠,通过PCR和免疫印迹(Western blot),确证OTUB1肝脏特异性基因敲除小鼠模型是否成功构建。通过组织病理学方法,分析主要组织器官的形态以及是否存在自发的病变;通过血清生化指标检测肝脏脂代谢水平;通过血糖耐受实验(GTT)分析HCKO小鼠对血糖的控制。结果:基因组测序和Western blot检测结果显示HCKO小鼠肝脏中OTUB1被敲除,其他组织中OTUB1表达水平无变化,证明OTUB1肝脏特异性基因敲除小鼠模型构建成功。HCKO小鼠出生正常,各组织器官无异常,生化指标中总胆固醇水平明显降低,表明OTUB1影响肝脏脂代谢水平。糖耐受实验中HCKO小鼠血糖回落迅速,表明敲除OTUB1影响肝脏血糖调节稳态。结论:应用Cre/Loxp技术成功建立OTUB1肝脏特异性基因敲除小鼠模型,为研究OTUB1在肝脏的生理功能和调控机制提供了重要的动物模型。     

海马皮质特异性敲除AEG-1基因小鼠的构建及其行为学初步研究*

星形胶质细胞上调基因-1(astrocyte upregulating gene-1,AEG-1)是HIV伴随老年痴呆患者脑组织中发现的星形胶质细胞上调基因之一,近年来研究表明其调控多种中枢神经系统疾病,但其在学习认知上的研究尚未见报道。海马和皮质在学习认知中起重要作用,利用CRISPR/Cas9技术结合Cre/loxp系统构建海马皮质特异性AEG-1敲除小鼠,在此模型鼠的基础上对AEG-1和学习认知的相关性进行初步研究。首先构建插入loxp位点的flox纯合型AEG-1^fl/fl小鼠,与海马、新皮层特异性表达Cre^+/+重组酶的工具鼠进行繁育,利用PCR技术筛选出子代基因型为AEG-1^fl/fl Cre⁺的海马皮质特异性AEG-1敲除小鼠;然后利用Western blot技术和免疫荧光技术检测AEG-1基因在小鼠海马皮质中的敲除效率;最后应用新物体识别箱和三腔社会互动箱并结合SMART 3.0分析系统,对海马皮质特异性AEG-1敲除小鼠的学习记忆和社会交互行为学进行初步评价。结果显示:成功获得子代基因型为AEG-1^fl/fl Cre⁺的基因敲除小鼠;AEG-1条件性敲除小鼠海马和皮质中AEG-1蛋白质表达水平较对照组显著降低;新物体识别结果表明AEG-1条件性敲除小鼠的区分系数明显低于对照组,表明AEG-1条件性敲除小鼠的学习记忆能力较弱,但是三腔交互结果表明AEG-1条件性敲除小鼠在社会交互上与对照组相比无明显差异。以上结果为AEG-1在学习认知方面的进一步研究奠定了基础。     

海马皮质特异性敲除AEG-1基因小鼠的构建及其行为学初步研究*

星形胶质细胞上调基因-1(astrocyte upregulating gene-1,AEG-1)是HIV伴随老年痴呆患者脑组织中发现的星形胶质细胞上调基因之一,近年来研究表明其调控多种中枢神经系统疾病,但其在学习认知上的研究尚未见报道。海马和皮质在学习认知中起重要作用,利用CRISPR/Cas9技术结合Cre/loxp系统构建海马皮质特异性AEG-1敲除小鼠,在此模型鼠的基础上对AEG-1和学习认知的相关性进行初步研究。首先构建插入loxp位点的flox纯合型AEG-1^fl/fl小鼠,与海马、新皮层特异性表达Cre^+/+重组酶的工具鼠进行繁育,利用PCR技术筛选出子代基因型为AEG-1^fl/fl Cre⁺的海马皮质特异性AEG-1敲除小鼠;然后利用Western blot技术和免疫荧光技术检测AEG-1基因在小鼠海马皮质中的敲除效率;最后应用新物体识别箱和三腔社会互动箱并结合SMART 3.0分析系统,对海马皮质特异性AEG-1敲除小鼠的学习记忆和社会交互行为学进行初步评价。结果显示:成功获得子代基因型为AEG-1^fl/fl Cre⁺的基因敲除小鼠;AEG-1条件性敲除小鼠海马和皮质中AEG-1蛋白质表达水平较对照组显著降低;新物体识别结果表明AEG-1条件性敲除小鼠的区分系数明显低于对照组,表明AEG-1条件性敲除小鼠的学习记忆能力较弱,但是三腔交互结果表明AEG-1条件性敲除小鼠在社会交互上与对照组相比无明显差异。以上结果为AEG-1在学习认知方面的进一步研究奠定了基础。     

Hepatocyte-Specific Arid1a Deficiency Initiates Mouse Steatohepatitis and Hepatocellular Carcinoma

ARID1A, encoding a subunit of chromatin remodeling SWI/SNF complexes, has recently been considered as a new type of tumor suppressor gene for its somatic mutations frequently found in various human tumors, including hepatocellular carcinoma (HCC). However, the role and mechanism of inactivated ARID1A mutations in tumorigenesis remain unclear. To investigate the role of ARID1A inactivation in HCC pathogenesis, we generated hepatocyte-specific Arid1a knockout (Arid1a ^LKO) mice by crossing mice carrying loxP-flanked Arid1a exon 8 alleles (Arid1a ^f/f) with albumin promoter-Cre transgenic mice. Significantly, the hepatocyte-specific Arid1a deficiency results in mouse steatohepatitis and HCC development. In Arid1a ^LKO mice, we found that innate immune cells, including F4/80+ macrophages and CD11c+ neutrophil cells, infiltrate into the liver parenchyma, accompanied by the increased tumor necrosis factor (TNF)-α and interleukin (IL)-6, and activation of STAT3 and NF-κB pathways. In conclusion, hepatocyte-specific Arid1a deficiency could lead to mouse steatohepatitis and HCC development. This study provides an alternative mechanism by which Arid1a deficiency contributes to HCC tumorigenesis.     

Generation and basic characterization of a gene-trap knockout mouse model of Scn2a with a substantial reduction of voltage-gated sodium channel Nav1.2 expression

Large-scale genetic studies revealed SCN2A as one of the most frequently mutated genes in patients with neurodevelopmental disorders. SCN2A encodes for the voltage-gated sodium channel isoform 1.2 (Na_v1.2) expressed in the neurons of the central nervous system. Homozygous knockout (null) of Scn2a in mice is perinatal lethal, whereas heterozygous knockout of Scn2a (Scn2a^+/−) results in mild behavior abnormalities. The Na_v1.2 expression level in Scn2a^+/− mice is reported to be around 50–60% of the wild-type (WT) level, which indicates that a close to 50% reduction of Na_v1.2 expression may not be sufficient to lead to major behavioral phenotypes in mice. To overcome this barrier, we characterized a novel mouse model of severe Scn2a deficiency using a targeted gene-trap knockout (gtKO) strategy. This approach produces viable homozygous mice (Scn2a^gtKO/gtKO) that can survive to adulthood, with about a quarter of Na_v1.2 expression compared to WT mice. Innate behaviors like nesting and mating were profoundly disrupted in Scn2a^gtKO/gtKO mice. Notably, Scn2a^gtKO/gtKO mice have a significantly decreased center duration compared to WT in the open field test, suggesting anxiety-like behaviors in a novel, open space. These mice also have decreased thermal and cold tolerance. Additionally, Scn2a^gtKO/gtKO mice have increased fix-pattern exploration in the novel object exploration test and a slight increase in grooming, indicating a detectable level of repetitive behaviors. They bury little to no marbles and have decreased interaction with novel objects. These Scn2a gene-trap knockout mice thus provide a unique model to study pathophysiology associated with severe Scn2a deficiency.     

Deficiency of the BMP Type I receptor ALK3 partly protects mice from anemia of inflammation

Background

Inflammatory stimuli induce the hepatic iron regulatory hormone hepcidin, which contributes to anaemia of inflammation (AI). Hepcidin expression is regulated by the bone morphogenetic protein (BMP) and the interleukin-6 (IL-6) signalling pathways. Prior results indicate that the BMP type I receptor ALK3 is mainly involved in the acute inflammatory hepcidin induction four and 72 h after IL-6 administration. In this study, the role of ALK3 in a chronic model of inflammation was investigated. The intact, heat-killed bacterium Brucella abortus (BA) was used to analyse its effect on the development of inflammation and hypoferremia in mice with hepatocyte-specific Alk3-deficiency (Alk3^fl/fl; Alb-Cre) compared to control (Alk3^fl/fl) mice.

Results

An iron restricted diet prevented development of the iron overload phenotype in mice with hepatocyte-specific Alk3 deficiency. Regular diet leads to iron overload and increased haemoglobin levels in these mice, which protects from the development of AI per se. Fourteen days after BA injection Alk3^fl/fl; Alb-Cre mice presented milder anaemia (Hb 16.7 g/dl to 11.6 g/dl) compared to Alk3^fl/fl control mice (Hb 14.9 g/dl to 8.6 g/dl). BA injection led to an intact inflammatory response in all groups of mice. In Alk3^fl/fl; Alb-Cre mice, SMAD1/5/8 phosphorylation was reduced after BA as well as after infection with Staphylococcus aureus. The reduction of the SMAD1/5/8 signalling pathway due to hepatocyte-specific Alk3 deficiency partly suppressed the induction of STAT3 signalling.

Conclusion

The results reveal in vivo, that 1) hepatocyte-specific Alk3 deficiency partly protects from AI, 2) the development of hypoferremia is partly dependent on ALK3, and 3) the ALK3/BMP/hepcidin axis may serve as a possible therapeutic target to attenuate AI.

     

SPC-Cre-ERT2 Transgenic Mouse for Temporal Gene Deletion in Alveolar Epithelial Cells

Although several Cre-loxP-based gene knockout mouse models have been generated for the study of gene function in alveolar epithelia in the lung, their applications are still limited. In this study, we developed a SPC-Cre-ER^T2 mouse model, in which a tamoxifen-inducible Cre recombinase (Cre-ER^T2) is under the control of the human surfactant protein C (SPC) promoter. The specificity and efficiency of Cre-ER^T2 activity was first evaluated by crossing SPC-Cre-ER^T2 mouse with ROSA26R mouse, a β-galactosidase reporter strain. We found that Cre-ER^T2 was expressed in 30.7% type II alveolar epithelial cells of SPC-Cre-ER^T2/ROSA26R mouse lung tissues in the presence of tamoxifen. We then tested the tamoxifen-inducible recombinase activity of Cre-ER^T2 in a mouse strain bearing TSC1 conditional knockout alleles (TSC1^fx/fx). TSC1 deletion was detected in the lungs of tamoxifen treated SPC-Cre-ER^T2/TSC1^fx/fx mice. Therefore this SPC-Cre-ER^T2 mouse model may be a valuable tool to investigate functions of genes in lung development, physiology and disease.     

Knockout of the l-pgds gene aggravates obesity and atherosclerosis in mice

This study was designed to determine whether lipocalin type-prostaglandin D synthase (l-pgds) deficiency contributes to atherogenesis using gene knockout (KO) mice. A high-fat diet was given to 8-week-old C57BL/6 (wild type; WT), l-pgds KO (LKO), apolipoprotein E (apo E) KO (AKO) and l-pgds/apo E double KO (DKO) mice. The l-pgds deficient mice showed significantly increased body weight, which was accompanied by increased size of subcutaneous and visceral fat tissues. Fat deposition in the aortic wall induced by the high-fat diet was significantly increased in LKO mice compared with WT mice, although there was no significant difference between AKO and DKO mice. In LKO mice, atherosclerotic plaque in the aortic root was also increased and, furthermore, macrophage cellularity and the expression of pro-inflammatory cytokines such as interleukin-1β and monocyte chemoattractant protein-1 were significant increased. In conclusion, l-pgds deficiency induces obesity and facilitates atherosclerosis, probably through the regulation of inflammatory responses.     

Acquired Deficiency of A20 Results in Rapid Apoptosis,Systemic Inflammation,and Abnormal Hematopoietic Stem Cell Function

A20 is a negative regulator of NF-κB, and mutational loss of A20 expression is involved in the pathogenesis of autoimmune diseases and B-cell lymphomas. To clarify the role of A20 in adult hematopoiesis, we generated conditional A20 knockout mice (A20^flox/flox) and crossed them with Mx–1Cre (MxCre ⁺) and ERT2Cre (ERT2Cre ⁺) transgenic mice in which Cre is inducibly activated by endogenous interferon and exogenous tamoxifen, respectively. A20^flox/flox MxCre ⁺ (A20Mx) mice spontaneously exhibited myeloid proliferation, B cell apoptosis, and anemia with overproduction of pro-inflammatory cytokines. Bone marrow transplantation demonstrated that these changes were caused by hematopoietic cells. NF-κB was constitutively activated in A20Mx hematopoietic stem cells (HSCs), which caused enhanced cell cycle entry and impaired repopulating ability. Tamoxifen stimulation of A20^flox/flox ERT2Cre ⁺ (A20ERT2) mice induced fulminant apoptosis and subsequent myeloproliferation, lymphocytopenia, and progressive anemia with excessive production of pro-inflammatory cytokines, as observed in A20Mx mice. These results demonstrate that A20 plays essential roles in the homeostasis of adult hematopoiesis by preventing apoptosis and inflammation. Our findings provide insights into the mechanism underlying A20 dysfunction and human diseases in which A20 expression is impaired.     

Unchanged survival rates of Shadoo knockout mice after infection with mouse-adapted scrapie

Previous studies have demonstrated that Shadoo (Sho), a GPI-linked glycoprotein encoded by the Sprn gene with a membrane localization similar to PrP^C, is reduced in the brains of rodents with terminal prion disease. To determine the functional significance of Sho in prion disease pathogenesis, Sho-deficient mice were generated by gene targeting. Sho knockout and control wild-type (WT) mice were infected with themouse-adapted scrapie strains 22L or RML. No significant differences in survival, the incubation period of prion disease or other disease features were observed between Sho mutant and WT mice. In this model of prion disease, Sho removal had no effect on disease pathogenesis.     

Influence of knockout of <Emphasis Type="Italic">At4g20990</Emphasis> gene encoding α-CA4 on photosystem II light-harvesting antenna in plants grown under different light intensities and day lengths

Effect of knockout of the At4g20990 gene encoding α-carbonic anhydrase 4 (α-CA4) in Arabidopsis thaliana in plants grown in low light (LL, 80 μmol photons m^?2 s^?1) or in high light (HL, 400 μmol photons m^?2 s^?1) under long (LD, 16 h) or short (SD, 8 h) day length was studied. In α-CA4 knockout plants, under all studied conditions, the non-photochemical quenching was lower; the decrease was more pronounced under HL. This pointed to α-CA4 implication in the processes leading to energy dissipation in PSII antenna. In this context the content of major antenna proteins Lhcb1 and Lhcb2 was lower in α-CA4 knockouts than in wild-type (WT) plants under all growth conditions. The expression level of lhcb2 gene was also lower in mutants grown under LD, LL and HL in comparison to WT. At the same time, this level was higher in mutants grown under SD, LL and it was the same under SD, HL. Overall, the data showed that the knockout of the At4g20990 gene affected both the contents of proteins of PSII light-harvesting complex and the expression level of genes encoding these proteins, with peculiarities dependent on day length. These data together with the fact of a decrease of non-photochemical quenching of leaf chlorophyll a fluorescence in α-CA4-mut as compared with that in WT plants implied that α-CA4 participates in acclimation of photosynthetic apparatus to light intensity, possibly playing important role in the photoprotection. The role of this CA can be especially important in plants growing under high illumination conditions.