Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice

Two transgenic mice were obtained that contain in their chromosomes the complete hepatitis B virus (HBV) genome except for the core gene. These mice secrete particles of HBV surface antigen (HBsAg) in the serum. In one mouse, HBV DNA sequences that had integrated at two different sites were shown to segregate independently in the first filial generation (F1) and only one of the sequences allowed expression of the surface antigen. Among these animals the males produced five to ten times more HBsAg than the females. A 2.1-kilobase messenger RNA species comigrating with the major surface gene messenger RNA is expressed specifically in the liver in the two original mice. The results suggest that the HBV sequences introduced into the mice are able to confer a tissue-specific expression to the S gene. In addition, the HBV transgenic mice represent a new model for the chronic carrier state of hepatitis B virus infection.     

Synthesis of functional human hemoglobin in transgenic mice

Human alpha- and beta-globin genes were separately fused downstream of two erythroid-specific deoxyribonuclease (DNase) I super-hypersensitive sites that are normally located 50 kilobases upstream of the human beta-globin gene. These two constructs were coinjected into fertilized mouse eggs, and expression was analyzed in transgenic animals that developed. Mice that had intact copies of the transgenes expressed high levels of correctly initiated human alpha- and beta-globin messenger RNA specifically in erythroid tissue. An authentic human hemoglobin was formed in adult erythrocytes that when purified had an oxygen equilibrium curve identical to the curve of native human hemoglobin A (Hb A). Thus, functional human hemoglobin can be synthesized in transgenic mice. This provides a foundation for production of mouse models of human hemoglobinopathies such as sickle cell disease.     

Antisense RNA directed against the 3' noncoding region prevents dormant mRNA activation in mouse oocytes

Primary mouse oocytes contain untranslated stable messenger RNA for tissue plasminogen activator (t-PA). During meiotic maturation, this maternal mRNA undergoes a 3'-polyadenylation, is translated, and is degraded. Injections of maturing oocytes with different antisense RNA's complementary to both coding and noncoding portions of t-PA mRNA all selectively blocked t-PA synthesis. RNA blot analysis of t-PA mRNA in injected, matured oocytes suggested a cleavage of the RNA.RNA hybrid region, yielding a stable 5' portion, and an unstable 3' portion. In primary oocytes, the 3' noncoding region was susceptible to cleavage, while the other portions of the mRNA were blocked from hybrid formation until maturation occurred. Injection of antisense RNA complementary to 103 nucleotides of its extreme 3' untranslated region was sufficient to prevent the polyadenylation, translational activation, and destabilization of t-PA mRNA. These results demonstrate a critical role for the 3' noncoding region of a dormant mRNA in its translational recruitment during meiotic maturation of mouse oocytes.     

SPRN转基因小鼠感染Scraipe鼠适应性病毒后动物行为与脑组织形态比较

通过建立Scraipe鼠适应性病毒感染Tg(sprn-B)/C57BL/6转基因小鼠与C57BL/6野生型小鼠,观察鼠行为学及脑组织形态学的病理变化,探讨Shadoo蛋白在感染过程中对小鼠学习记忆能力及中枢神经系统的影响。结果显示在Morris水迷宫定位航行中,4~7 d转基因感染组与野生型感染组的逃避潜伏期差异极显著(P<0.01);空间探索试验中,转基因感染组与野生型感染组的第1次穿越平台时间及穿越平台次数差异极显著(P<0.01)。HE染色观察鼠脑切片发现,转基因感染组与野生型感染组相比,海马、皮质、小脑空泡数量略少,海马区锥体层细胞排列比较紧密,细胞轮廓比较完整,小脑蒲肯野氏细胞清晰明显。     

Expression of functional cell-cell channels from cloned rat liver gap junction complementary DNA

An oocyte expression system was used to test the relation between a complementary DNA (cDNA) clone encoding the liver gap junction protein and cell-cell channels. Total liver polyadenylated messenger RNA injected into oocytes induced cell-cell channels between paired oocytes. This induction was blocked by simultaneous injection of antisense RNA transcribed from the gap junction cDNA. Messenger RNA selected by hybridization to the cDNA clone and translated in oocyte pairs yielded a higher junctional conductance than unselected liver messenger RNA. Cell-cell channels between oocytes were also formed when the cloned cDNA was expressed under the control of a heat-shock promoter. A concentration-dependent induction of channels was observed in response to injection with in vitro transcribed gap junction messenger RNA. Thus, the liver gap junction cDNA encodes a protein that is essential for the formation of functional cell-cell channels.     

G1/S transition in normal human T-lymphocytes requires the nuclear protein encoded by c-myb

Exposure of peripheral blood mononuclear cells (PBMC) to an 18-base c-myb antisense oligomer before mitogen or antigen stimulation resulted in almost complete inhibition of c-myb messenger RNA and protein synthesis and blockade of T lymphocyte proliferation. Expression of early and late activation markers, interleukin-2 receptor and transferrin receptor, respectively, by PBMC was unaffected by antisense oligomer exposure as was the expression of c-myc messenger RNA. In contrast, histone H3 messenger RNA levels and DNA content were selectively decreased. These results suggest that c-myb protein deprivation does not perturb T lymphocyte activation or early molecular events that may prepare the cell for subsequent proliferation. Rather, it appears to specifically block cells in late G1 or early S phase of the cell cycle.     

Viral persistence in neurons explained by lack of major histocompatibility class I expression

Viruses frequently persist in neurons, suggesting that these cells can evade immune surveillance. In a mouse model, 5 x 10(6) cytotoxic T lymphocytes (CTLs), specific for lymphocytic choriomeningitis virus (LCMV), did not lyse infected neurons or cause immunopathologic injury. In contrast, intracerebral injection of less than 10(3) CTL caused disease and death when viral antigens were expressed on leptomeningeal and choroid plexus cells of the nervous system. The neuronal cell line OBL21 expresses little or no major histocompatibility (MHC) class I surface glycoproteins and when infected with LCMV, resisted lysis by virus-specific CTLs. Expression of MHC heavy chain messenger RNA was limited, but beta 2-microglobulin messenger RNA and protein was made normally. OBL21 cells were made sensitive to CTL lysis by transfection with a fusion gene encoding another MHC class I molecule. Hence, neuronal cells probably evade immune surveillance by failing to express MHC class I molecules.     

Localization of amyloid beta protein messenger RNA in brains from patients with Alzheimer's disease

The distribution of cells containing messenger RNA that encodes amyloid beta protein was determined in hippocampi and in various cortical regions from cynomolgus monkeys, normal humans, and patients with Alzheimer's disease by in situ hybridization. Both 35S-labeled RNA antisense and sense probes to amyloid beta protein messenger RNA were used to ensure specific hybridization. Messenger RNA for amyloid beta protein was expressed in a subset of neurons in the prefrontal cortex from monkeys, normal humans, and patients with Alzheimer's disease. This messenger RNA was also present in the neurons of all the hippocampal fields from monkeys, normal humans and, although to a lesser extent in cornu ammonis 1, patients with Alzheimer's disease. The distribution of amyloid beta protein messenger RNA was similar to that of the neurofibrillary tangles of Alzheimer's disease in some regions, but the messenger RNA was also expressed in other neurons that are not usually involved in the pathology of Alzheimer's disease.     

拟南芥AtSEC14基因反义RNA植株的获得及抗病性检测

为确定拟南芥抗灰霉病相关基因AtSEC14的功能,本试验构建了AtSEC14基因的反义RNA载体;通过农杆菌介导的遗传转化方法,将其转化拟南芥野生型Col-0中;利用潮霉素抗性筛选和PCR检测,获得了阳性转基因植株。利用半定量RT-PCR技术,在转基因株系中未检测到AtSEC14基因的表达,说明该基因反义RNA载体的转入能特异影响AtSEC14基因的表达,表明试验所获得的转基因植株是AtSEC14基因的反义RNA转基因植株。对所获得的反义RNA转基因植株进行抗病性鉴定,发现反义RNA转基因植株对灰葡萄孢的敏感性增强,表明AtSEC14基因在拟南芥抗灰葡萄孢过程中起正调控的作用。     

Pancreatic neoplasia induced by SV40 T-antigen expression in acinar cells of transgenic mice

Three lines of transgenic mice were produced that develop pancreatic neoplasms as a consequence of expression of an elastase I-SV40 T-antigen fusion gene in the acinar cells. A developmental analysis suggests at least a two-stage process in the ontogeny of this disease. The first stage is a T antigen-induced, preneoplastic state characterized by a progression from hyperplasia to dysplasia of the exocrine pancreas, by an increased percentage of tetraploid cells, and by an arrest in acinar cell differentiation. The second stage is characterized by the formation of tumor nodules that appear to be monoclonal, because they have discrete aneuploid DNA contents. The cells within the nodules as compared to normal pancreatic tissue have less total RNA by a factor of 5, less pancreas-specific messenger RNA by a factor of about 50, and increased levels of T-antigen messenger RNA. A tumor cell line has been derived that retains both pancreatic and neoplastic properties.     

Identification of a peptide specific for Aplysia sensory neurons by PCR-based differential screening.

In order to identify genes specific for the sensory neurons of Aplysia, a miniaturized differential screening method based on the polymerase chain reaction and applicable to small amounts of tissue was used. One messenger RNA was isolated that is expressed in every mechanoreceptor sensory cluster of the Aplysia central nervous system. This messenger RNA encodes a peptide that seems to function as an inhibitory cotransmitter. The peptide selectively inhibits certain postsynaptic cells but not others and thereby allows the sensory neurons to achieve target-specific synaptic actions.     

Brain region and gene specificity of neuropeptide gene expression in cultured astrocytes

Astrocytes have many neuronal characteristics, such as neurotransmitter receptors, ion channels, and neurotransmitter uptake systems. Cultured astrocytes were shown to express certain neuropeptide genes, with specificity for both the gene expressed and the brain region from which the cells were prepared. Somatostatin messenger RNA and peptides were detected only in cerebellar astrocytes, whereas proenkephalin messenger RNA and enkephalin peptides were present in astrocytes of cortex, cerebellum, and striatum. Cholecystokinin was not expressed in any of the cells. These results support the hypothesis that peptides synthesized in astrocytes may play a role in the development of the central nervous system.     

BMP4对小鼠毛囊数量的影响

利用PolⅡ型人角蛋白14基因的启动子K14驱动eGFP-shRNA整合转录本的表达方法,制备在皮肤组织中高效表达靶向BMP4基因的siRNA转基因小鼠模型。利用Northern杂交的方法验证siRNA在转基因小鼠皮肤中高表达,但未从mRNA水平分析BMP4基因的表达变化。以建立的转基因小鼠模型为基础,检测小鼠皮肤组织中BMP4基因的表达变化,并观察妊娠18.5d(E18.5)转基因胎鼠与同窝阴性胎鼠背部皮肤组织切片中毛囊数量的变化,选择5个不同的视野进行毛囊数量计数后统计分析。结果表明,转基因小鼠皮肤组织中BMP4基因的表达下调,同时毛囊数量显著增加。表明利用融合表达载体制备转基因小鼠实现目的基因RNAi的方法可以有效地抑制动物体内目的基因的表达,同时也从胎鼠毛囊数量分析统计的结果发现,该基因对小鼠毛囊的发生发育有一定的影响。     

Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin mu

Antibody-producing cells display a special form of regulation whereby each cell produces immunoglobulin from only one of its two sets of antibody genes. This phenomenon, called allelic exclusion, is thought to be mediated by the product of one heavy chain allele restricting the expression of the other. Heavy chains are synthesized in two molecular forms, secreted and membrane bound. In order to determine whether it is specifically the membrane-bound form of the immunoglobulin M (IgM) heavy chain (mu) that mediates this regulation, transgenic mice were created that carry a human mu chain gene altered so that it can only direct the synthesis of the membrane-bound protein. The membrane-bound form of the human mu chain was made by most of the B cells in these animals as measured by assays of messenger RNA and surface immunoglobulins. Further, the many B cells that express the human gene do not express endogenous mouse IgM, and the few B cells that express endogenous mouse mu do not express the transgene. Thus, the membrane-bound form of the mu chain is sufficient to mediate allelic exclusion. In addition, the molecular structures recognized for this purpose are conserved between human and mouse systems.     

Antisense RNA inactivation of myosin heavy chain gene expression in Dictyostelium discoideum

The role of myosin in the contraction of striated muscle cells is well known, but its importance in nonmuscle cells is not yet clear. The function of myosin in Dictyostelium discoideum has been investigated by isolating cells which specifically lack myosin heavy chain (MHC A) protein. Cells were transformed with a vector encoding RNA complementary to mhcA messenger RNA (antisense RNA). Stable transformants have a dramatic reduction in the amount of MHC A protein, grow slowly, and generate giant multinucleated progeny, indicating an impairment in cytokinesis. Surprisingly, the cells adhere to surfaces, extend pseudopods and are capable of ameboid locomotion. The developmental sequence that is initiated by starving cells is severely impaired by the lack of myosin. The cells are unable to form multicellular aggregates normally and do not undergo subsequent morphogenesis. By changing the food source from liquid medium to bacteria, expression of the endogenous mhcA messenger RNA can be increased relative to expression of antisense RNA. When grown in this way, the transformed cells accumulate MHC A protein, remain mononucleate, and proceed through development normally.     

Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping

Most mutations in the dystrophin gene create a frameshift or a stop in the mRNA and are associated with severe Duchenne muscular dystrophy. Exon skipping that naturally occurs at low frequency sometimes eliminates the mutation and leads to the production of a rescued protein. We have achieved persistent exon skipping that removes the mutated exon on the dystrophin messenger mRNA of the mdx mouse, by a single administration of an AAV vector expressing antisense sequences linked to a modified U7 small nuclear RNA. We report the sustained production of functional dystrophin at physiological levels in entire groups of muscles and the correction of the muscular dystrophy.     

A chicken transferrin gene in transgenic mice escapes X-chromosome inactivation

Mammalian X-chromosome inactivation involves a coordinate shutting down of physically linked genes. Several proposed models require the presence of specific sequences near genes to permit the spread of inactivation into these regions. If such models are correct, one might predict that heterologous genes transferred onto the X chromosome might lack the appropriate signal sequences and therefore escape inactivation. To determine whether a foreign gene inserted into the X chromosome is subject to inactivation, transgenic mice harboring 11 copies of the complete, 17-kilobase chicken transferrin gene on the X chromosome were used. Male mice hemizygous for this insert were bred with females bearing Searle's translocation, an X-chromosome rearrangement that is always active in heterozygous females (the unrearranged X chromosome is inactive). Female offspring bearing the Searle's translocation and the chicken transferrin gene had the same amount of chicken transferrin messenger RNA in liver as did transgenic male mice or transgenic female mice lacking the Searle's chromosome. This result shows that the inserted gene is not subject to X-chromosome inactivation and suggests that the inactivation process cannot spread over 187 kilobases of DNA in the absence of specific signal sequences required for inactivation.     

水稻蜡质合成相关基因 OsCER4 自身启动子驱动的反义RNA转化植株获得

水稻OsCER4基因编码脂肪醛脱羧酶,与拟南芥CER1基因高度同源,是否参与植物表面蜡质合成尚不清楚,为了探讨其功能,扩增了OsCER4基因起始密码子ATG上游约2 kb的序列作为该基因的启动子,以常规技术将启动子和OsCER4基因反义片段克隆到pCAMBIA双元载体1380中,采用农杆菌介导法转化粳稻品种中花11,并进行了转化苗OsCER4蛋白的表达量变化分析.结果表明,成功构建了由OsCER4自身启动子驱动的反义RNA载体,并通过农杆菌介导法成功转入水稻中花11中,多数OsCER4基因反义转化植株为阳性转化植株,后代分离比符合3∶1,反义RNA转化植株在蛋白水平上的表达量下降.