RPS14基因重组慢病毒载体的构建及其在MUTZ-8细胞中的表达

目的构建携带人RPS14基因重组慢病毒载体获得稳定产毒的细胞,并观察其在人MDS细胞株MUTZ-8细胞中的表达。方法通过反转录方法,获得目的基因RPS14;将目的基因克隆入慢病毒表达质粒pGC-FU Vector,构建含有RPS14基因的重组慢病毒载体pGC-FU RPS14,经PCR、酶切及测序鉴定其正确性;在Lipofectamine 2000介导下,将成功构建的质粒pGC-FU RPS14与慢病毒包装质粒pHelper 1.0、pHelper 2.0共转染293T细胞,通过实时定量PCR法测定病毒滴度,并将获得的重组慢病毒载体pGC-FU RPS14转染人MDS细胞株MUTZ-8,用流式细胞仪检测转染效率,RT-PCR检测靶细胞内RPS14 mRNA的表达。结果含有RPS14基因的重组慢病毒载体经PCR、酶切和测序鉴定构建正确,且测定的病毒滴度为2.0×109 TU/ml。流式细胞仪检测其转染效率为68.41%,RT-PCR检测到转染后RPS14 mRNA在靶细胞中的稳定表达。结论构建了携带人RPS14基因的慢病毒载体,转染人MUTZ-8细胞株后能够稳定表达RPS14基因。

拟南芥RPS14基因的克隆表达及功能初步研究

拟南芥RPS14(Ribosomal protein S14)是从拟南芥中分离出的一种核糖体蛋白,是核糖体40S亚基的组成成分,属于S11P核糖体蛋白家族,主要负责RNA转录后加工。提取拟南芥总RNA,用RT-PCR技术得到RPS14全长基因,经T-A克隆插入到pEASY-T3载体上。利用亚克隆法成功构建pGEX-6P-1-RPS14原核表达质粒。GST-RPS14融合蛋白在大肠杆菌BL21(DE3)中表达,分子量约为43 kD。运用同样方法构建pET-28a-AK6原核表达质粒,获得大小约为26 kD的HIS-AK6融合蛋白。运用体外pull-down技术,并用SDS-PAGE和Western blot进行验证,证明拟南芥RPS14蛋白与AK6蛋白之间存在相互作用。因此,推测拟南芥的AK6与RPS14蛋白共同参与核酸代谢过程,影响RNA转录后加工,进而抑制拟南芥茎细胞的生长,使植株表现矮小特征。

RPS14基因RNAi慢病毒载体构建及在SKM-1细胞中的表达

背景:有研究表明,造血干/祖细胞内RPS14表达减少可导致造血细胞病态造血,尤其是向红系分化受阻,提示RPS14的正常表达有助于维持机体的正常造血。目的:构建RPS14基因RNAi慢病毒载体,并观察该基因在人骨髓增生异常综合征细胞株SKM-1细胞中的表达。方法:针对已经筛选确定的RPS14基因RNAi有效靶序列,构建GC-shRPS14慢病毒载体并测序鉴定。用GC-shRPS14、pHelper1.0载体和pHelper2.0载体共转染包装细胞293T细胞,包装产生慢病毒,测定病毒滴度;并将获得的RPS14shRNA慢病毒载体GC-shRPS14转染人骨髓增生异常综合征细胞株SKM-1,用Western Blot检测靶细胞内RPS14蛋白的表达。结果与结论:经测序证实,构建出了RPS14shRNA的慢病毒载体GC-shRPS14。包装慢病毒,浓缩病毒悬液的滴度为2×109TU/mL。荧光显微镜下能直接观察到转染组细胞的GFP表达,转染效率为75%,WesternBlot检测到转染后RPS14在靶细胞中表达明显被抑制。说明成功构建RPS14基因RNAi慢病毒载体,转染人SKM-1细胞株后RPS14蛋白表达沉默。

The overexpression of Rps14 in Lgr5+progenitor cells promotes hair cell regeneration in the postnatal mouse cochlea

Sensory hair cells(HCs)in the cochlea cannot regenerate spontaneously in adult mammals after being damaged by external or genetic factors.However,several genes and signaling pathways are reported to induce cochlear HC regeneration at the early neonatal stage.Rps14 encodes a ribosomal protein that is involved in the regulation of cell differentiation and proliferation in mammals.However,its roles in the cochlea have not been reported in vivo.Here,we specifically overexpressed Rps14 in Lgr5+progenitor cells in the newborn mouse cochlea and found that Rps14 conditional overexpression(cOE)mice had significantly increased the ectopic HCs,including inner and outer HCs.We further explored the source of these ectopic HCs and found no EdU+supporting cells observed in the Rps14 cOE mice.The lineage tracing results,on the other hand,revealed that Rps14 cOE mice had significantly more tdTomato+HCs in their cochleae than control mice.These results indicated that regenerated HCs by cOE of Rps14 are most likely derived from inducing the direct trans-differentiation of Lgr5+progenitor cells into HCs.Moreover,real-time qPCR results suggested that the transcription factor genes Atoh1 and Gfi1,which are important in regulating HC differentiation,were upregulated in the cochlear basilar membrane of Rps14 cOE mice.In summary,this study provides in vivo evidence that,in the postnatal mouse cochlea,Rps14 is a potential gene that can promote the spontaneous trans-differentiation of Lgr5+progenitor cells into HCs.This gene may one day be exploited as a therapeutic target for treating hearing loss.

玉米RPS14 cDNA的克隆及序列分析

为了研究玉米RPS14基因的序列信息,以玉米‘南-22号’为实验材料,根据已报道的部分核糖体蛋白S14亚基基因的相关信息,利用RT-PCR克隆出玉米RPS14基因的cDNA序列,并对序列进行了分析。结果表明,玉米RPS14基因的开放性阅读框(ORF)长453 bp,编码150个氨基酸,GenBank登陆号为JX232265。蛋白质的相对分子量约为16.16 kD,等电点pI为11.35。该蛋白含有7个不同功能位点。该基因的cDNA序列及其编码的氨基酸序列,与已报道的部分植物RPS14基因有很高的同源性。本研究为进一步研究RPS14基因的功能提供了基础。

A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure

5q-syndrome is a distinct form of myelodysplastic syndrome（MDS） where a deletion on chromosome 5 is the underlying cause.MDS is characterized by bone marrow failures,including macrocytic anemia.Genetic mapping and studies using various models support the notion that ribosomal protein S14（RPS14） is the candidate gene for the erythroid failure.Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis,similar to what is observed with other ribosomal proteins.However,due to the higher risk for cancer development in patients with ribosome deficiency,targeting the p53 pathway is not a viable treatment option.To better understand the pathology of RPS14 deficiency in 5q-deletion,we generated a zebrafish model harboring a mutation in the RPS14 gene.This model mirrors the anemic phenotype seen in 5q-syndrome.Moreover,the anemia is due to a late-stage erythropoietic defect,where the erythropoietic defect is initially p53-independent and then becomes p53-dependent.Finally,we demonstrate the versatility of this model to test various pharmacological agents,such as RAP-011,L-leucine,and dexamethasone in order to identify molecules that can reverse the anemic phenotype.

p53在5q-综合征发病机制中的研究现状

5q-综合征是骨髓增生异常综合征(MDS)的特殊亚型,该病患者的临床表现以贫血为主,血小板计数可正常或增高,骨髓原始细胞比例<5%,预后较好。随着对5q-综合征研究的进展,其发病机制逐渐被揭示。位于5号染色体长臂(5q)的CSNK1A1、RPS14、EGR1等致病基因单倍剂量不足,是引起该病不同临床表型的主要分子机制,其中多个基因的缺失与TP53的表达产物p53相关。此外,伴5号染色体异常MDS患者的TP53突变率较MDS总体人群明显升高,因此p53在5q-综合征的发病机制中发挥重要作用,并已成为其潜在治疗靶点。目前,来那度胺为5q-综合征的一线用药,其作用机制也与p53相关。笔者拟就RPS14、CSNK1A1、EGR1等关键致病基因单倍剂量不足、TP53突变通过p53参与5q-综合征的发病,以及来那度胺通过p53对5q-综合征发挥治疗作用等方面,对p53在5q-综合征发病机制中的研究现状进行阐述。