人Man2c1转基因小鼠模型的建立

目的 为在体内研究MAN2C1的生物学意义而建立转hMan2c1基因的小鼠。方法 构建pIRKS2-EGFP-hMan2c1重组表达载体，经体外转染实验鉴定转染的基因能在COS-7细胞表达后，注射人ICR小鼠受精卵，以制备转基因小鼠。用基因组PCR鉴定目的基因在宿主基因组DNA的整合。用RT-PCR和Westernblot分析hMan2c1在转基因小鼠的表达。结果 在116只原代小鼠中，有7只hMan2c1基因组PCR阳性。在所检测的20只F1代小鼠中，有9只hMan2c1基因组PCR阳性。在所检测的21只F2代小鼠中，有16只基因组PCR阳性。用鼠尾组织RT-PCR和Western blot检测hMan2c1基因表达，确定基因组PCR阳性的7个系中有4个系阳性。结论 建立了4个稳定表达hMan2c1的转基因小鼠系，为深入研究MAN2C1的生物学意义打下了基础。     

寻常型银屑病患者皮损中HBD-2和TNF-α的表达及意义

目的:探讨寻常型银屑病皮损中人β防御素2(HBD-2)与肿瘤坏死因子α(TNF-α)的表达及其相关性。方法:采用实时荧光定量逆转录聚合酶链反应法(RT-PCR)检测45例寻常型银屑病患者的皮损组织、非皮损组织以及15例正常人皮肤组织中HBD-2 mRNA及TNF-αmRNA的表达。结果:寻常型银屑病皮损组HBD-2 mRNA和TNF-αmRNA表达显著高于非皮损组和正常对照组。在皮损组HBD-2 mRNA与TNF-αmRNA的表达呈正相关,且二者的表达均与患者PASI评分成正相关。结论:HBD-2和TNF-α在寻常型银屑病发病中可能起相互协同的作用,加重了银屑病的炎症反应。     

人β-防御素2联合乙肝疫苗对小鼠免疫效应的研究

目的:探讨人β-防御素2加强乙肝疫苗对小鼠免疫应答的作用。方法:采用乙肝疫苗2μg/次/只联合HBD2基因真核表达质粒pEGF-C1/HBD2 100μg/次/只,按间隔2周、共免疫3次的左后肢股四头肌肌肉接种方案免疫BALB/c小鼠,同时设立相应的对照。免疫后用双抗原夹心ELISA法测小鼠血清HBsAb水平。采用流式细胞仪检测脾脏CD4+T和CD8+T细胞百分率及CD4+/CD8+比值。应用CCK8试剂盒检测脾淋巴细胞在不同抗原刺激下的增殖反应。结果:pEGF-C1/HBD2质粒联合乙肝疫苗免疫小鼠抗体水平明显升高,CD4+T细胞百分率比其他免疫组都高,脾淋巴细胞体外增殖实验明显。与其他各免疫组相比,pEGF-C1空质粒联合乙肝疫苗免疫组抗体水平和淋巴细胞体外增殖情况及CD4+T细胞百分率都较低。结论:人β-防御素-2对乙肝疫苗在小鼠中的免疫功能有一定增强作用。     

应用显微注射法建立转基因小鼠

应用显微注射法建立转基因小鼠谢卫兵陈汉源曾位森罗琛（第一军医大学组胚教研室广州５１０５１５）转基因动物是研究基因表达调控及表达产物生物学效应的最佳体系之一，同时作为一种新型的生物反应器，在医学、农、林、畜牧业等领域有着广泛的应用前景。目前采用基因转移...     

LPS介导滋养层细胞TLR4信号传导通路对HBD-2表达的影响

目的研究LPS体外刺激滋养层细胞是否诱导表达HBD-2,并进一步探讨该表达与TLR4信号传导通路的关系。方法建立不同孕周的滋养层细胞原代培养体系,应用TLR4阻断剂预处理滋养层细胞30 min前后,给予不同质量浓度的LPS(25、50、100、200、400 ng/ml)作用72 h,采用SYBR GreenⅠ实时荧光定量RT-PCR技术检测滋养层细胞HBD-2 mRNA的表达。结果 1)LPS能诱导滋养层细胞HBD-2 mRNA表达,且这种表达与其具有浓度和时间依赖性;当质量浓度为200 ng/ml,刺激24 h时,HBD-2 mRNA的相对表达量最高;2)TLR4阻断剂能抑制LPS对滋养层细胞表达HBD-2 mRNA的诱导作用,与未阻断之前相比差异具有统计学意义(P<0.01)。结论 LPS可能通过TLR4信号传导通路诱导滋养层细胞表达HBD-2 mRNA,将为宫内感染防治提供新靶点。     

心肌组织特异性高表达核仁素转基因小鼠的构建与鉴定

 目的：构建并鉴定心肌组织特异性高表达核仁素(Ncl)的转基因小鼠,为从整体水平上研究核仁素对心肌功能的影响及其发挥心肌保护作用的机制提供动物模型。方法：采用基因重组法将小鼠核仁素基因的全长cDNA插入含心肌细胞特异性启动子的Alpha-MyHC clone 26载体,构建Alpha-MyHC clone 26-Ncl重组载体,通过双酶切及基因测序鉴定其正确性;采用PCR鉴定阳性小鼠并用Western blotting法观察转基因小鼠中核仁素的表达情况,采用组织切片及HE染色法观察转基因小鼠心肌组织形态;测量心脏重量指数和左室压最大上升速率,观察核仁素在心肌组织高表达后对小鼠心脏形态功能是否有影响。结果：PCR法观察显示获得4只阳性小鼠(51、52、56和86,其中52和86系繁殖良好）,Western blotting结果表明转基因小鼠的核仁素表达量明显高于野生鼠,HE染色等结果表明核仁素对心肌形态学、心脏/体重比值及心功能无明显影响。结论：成功制备了心肌组织特异性高表达核仁素的转基因小鼠,与野生型小鼠相比,转基因小鼠心肌形态及心功能无明显差异。     

MagA转基因小鼠模型的建立

目的建立MagA转基因小鼠,为活体MRI成像系统提供重要的实验动物模型。方法采用显微注射法．将MagA基因导入小鼠受精卵,再培育成转基因小鼠并繁殖传代。利用PCR方法鉴定转基因首建鼠及其子代基因型。结果酶切和DNA测序分析证实MagA基因准确克隆表达载体设计位点,目的基因序列与GenBank中MagA序列完全一致。通过PCR分析,进行转基因整合检测。目前转基因小鼠已稳定传代至第5代。结论MagA基因在转基因小鼠中整合,成功建立了MagA转基因小鼠。MagA转基因小鼠将成为MRI影像系统的重要实验动物模型。     

双重引物PCR鉴定APPSWE转基因小鼠

本实验旨在探讨一种简单易行,能有效识别APPSWE转基因小鼠基因检测中假阴性结果的方法。在PCR体系中设计两对引物,一对为APP基因特异性引物,另一对为根据其内源性管家基因β-actin设置的参照引物。利用同一个PCR反应体系,对两对引物进行扩增。结果显示,双重引物PCR扩增的特异性片段与单引物扩增片段完全吻合,利用该法检出的转基因阳性率高于单引物PCR检出的转基因阳性率。本方法简单易行,能够有效识别以往利用单一PCR检测出现的假阴性结果,并且具有很好的特异性和灵敏性,值得在转基因小鼠鉴定实验中推广。     

人ACE2转基因小鼠SARS-CoV感染后的病理变化及免疫学反应

目的 观察SARS-Cov感染人血管紧张素转换酶2(hACE2)转基因小鼠引起的病理变化并初步探讨其发生的免疫学机制,为SARS研究提供可靠的动物模型.方法 实时PCR测定hACE2转基因小鼠的拷贝数;用PUMC01株SARS-CoV感染hACE2转基因小鼠,光镜下观察小鼠全身组织器官的病理变化;ELISA方法检测血清特异性抗体和肺组织匀浆上清细胞因子TNF-a、IL-6、IFN-γ变化.结果 单拷贝hACE2转基因小鼠在感染SARS-CoV后肺组织出现更严重的间质性肺炎并伴有肺外多器官损伤;少数转基因小鼠血清检测到特异性IgC,抗体;转基因小鼠肺组织匀浆上清TNF-a、IL-6、IFN-γ的水平明显升高.结论 hACE2转基因小鼠感染SARS-CoV后出现与人类SARS患者相似的病理特征和免疫学反应,为研究SARS发病机制和药物评价提供了小动物模型.     

β-蜕皮甾酮对APP/PS-1转基因小鼠学习记忆能力及海马Bcl-2、Bax蛋白表达的影响

目的观察β-蜕皮甾酮对APP/PS-1转基因小鼠学习记忆能力及海马Bcl-2、Bax蛋白表达的影响。方法取C57小鼠6只及APP/PS-1转基因小鼠12只,C57小鼠为对照组(Control)、APP/PS-1小鼠12只随机分为模型组(APP/PS-1)和实验组(APP/PS-1+20HE),实验组APP/PS-1转基因小鼠按14.4mg/kg的剂量灌胃β-蜕皮甾酮,每日一次,对照组及模型组小鼠灌胃等量蒸馏水,连续灌胃8周,Morris水迷宫检测小鼠学习记忆能力,尼氏染色观察各组小鼠海马组织病理学变化,免疫组化法观察海马Bcl-2和Bax蛋白表达的变化。结果模型组小鼠与对照组小鼠相比,认知能力明显下降(P<0.01),海马Bcl-2和Bax蛋白表达均增加。实验组与模型组相比,学习记忆能力明显改善(P<0.01),Bcl-2蛋白的表达增加,Bax蛋白的表达减少。结论β-蜕皮甾酮能够改善转基因小鼠的学习记忆能力,可能与促进海马Bcl-2蛋白表达和抑制Bax蛋白表达有关。     

应用接头PCR克隆慢病毒介导的转基因小鼠整合位点旁侧序列

目的 为鉴定慢病毒介导的转基因小鼠中外源基因的整合位点信息,应用接头PCR克隆整合位点旁侧序列.方法 小鼠基因组总DNA酶解后与设计的接头片段连接,根据慢病毒的LTR序列设计巢式PCR引物,克隆转基因小鼠整合位点旁侧序列.结果 成功克隆到转基因小鼠整合位点的旁侧序列,经过测序定位于小鼠染色体上.结论 作为反向PCR的改进,本方法可用于转基因小鼠整合位点旁侧序列的克隆, 为分析整合位点与外源基因表达之间的关系等提供了科学依据.     

Francisella tularensis T-Cell Antigen Identification Using Humanized HLA-DR4 Transgenic Mice

There is no licensed vaccine against the intracellular pathogen Francisella tularensis. The use of conventional mouse strains to screen protective vaccine antigens may be problematic, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human antigen-presenting cells. We used engineered humanized mice that lack endogenous MHC class II alleles but that express a human HLA allele (HLA-DR4 transgenic [tg] mice) to identify potential subunit vaccine candidates. Specifically, we applied a biochemical and immunological screening approach with bioinformatics to select putative F. tularensis subsp. novicida T-cell-reactive antigens using humanized HLA-DR4 tg mice. Cell wall- and membrane-associated proteins were extracted with Triton X-114 detergent and were separated by fractionation with a Rotofor apparatus and whole-gel elution. A series of proteins were identified from fractions that stimulated antigen-specific gamma interferon (IFN-γ) production, and these were further downselected by the use of bioinformatics and HLA-DR4 binding algorithms. We further examined the validity of this combinatorial approach with one of the identified proteins, a 19-kDa Francisella tularensis outer membrane protein (designated Francisella outer membrane protein B [FopB]; FTN_0119). FopB was shown to be a T-cell antigen by a specific IFN-γ recall assay with purified CD4⁺ T cells from F. tularensis subsp. novicida ΔiglC-primed HLA-DR4 tg mice and cells of a human B-cell line expressing HLA-DR4 (DRB1*0401) functioning as antigen-presenting cells. Intranasal immunization of HLA-DR4 tg mice with the single antigen FopB conferred significant protection against lethal pulmonary challenge with an F. tularensis subsp. holarctica live vaccine strain. These results demonstrate the value of combining functional biochemical and immunological screening with humanized HLA-DR4 tg mice to map HLA-DR4-restricted Francisella CD4⁺ T-cell epitopes.Francisella tularensis is a Gram-negative bacterium and the etiological agent of the zoonotic disease tularemia. F. tularensis is classified into four subspecies, namely, F. tularensis subsp. tularensis, F. tularensis subsp. holarctica, F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida (F. novicida) on the basis of their biochemical and genetic profiles, virulence properties, and geographical origins (51). To this end, F. tularensis subsp. tularensis (type A) is the most virulent subspecies, with the inhalation of as few as 10 organisms causing disease and mortality rates of between 30 and 60% in untreated cases of pneumonic tularemia (53). The live vaccine strain (LVS) derived from F. tularensis subsp. holarctica has been used as a prophylactic vaccine against tularemia (48). Millions of individuals in the Soviet Union were immunized with live vaccine strains between 1946 and 1960 (52). However, LVS has not been licensed for use in the United States due to a lack of understanding of the genetic mutations that are responsible for attenuation of this strain, although it is used as an investigational new drug (IND) to immunize at-risk workers, primarily tularemia researchers. F. novicida, which causes disease only in immunocompromised humans but which is highly virulent for mice, has been used as a comparative model organism due to the high degree of genetic similarity with type A strains (98.1% homology between sequences common to strains U112 and SCHU S4 [45]). We recently reported that a defined vaccine strain (ΔiglB) generated in strain U112 was effective in inducing heterologous protection against various Francisella strains in a mouse model of pulmonary tularemia, suggesting the conservation of protective antigens (12).Cell-mediated immunity has been documented to play an important role in protection against tularemia (2, 18, 19, 49, 56). The role of antibodies, via neutralization and Fc receptor-mediated clearance (43, 44) in response to infection, has also gained significant attention. Therefore, the availability of a combination of multiple Francisella antigens containing T-cell and/or B-cell epitopes would be desirable for formulating an effective multivalent vaccine against this organism. However, the use of conventional mouse strains to identify protective antigens may not be feasible, given the differences in the major histocompatibility complex (MHC) binding properties between murine and human MHCs. These constraints can be overcome with the use of engineered humanized mice, such as the HLA-DR4 transgenic (tg) mouse. This mouse was generated to express the extracellular human α1 and β1 domains of the HLA-DRA and HLA-DRB1*0401 haplotypes, which form the peptide binding sites for antigen presentation, in conjunction with the murine α2 and β2 domains (29). These chimeric molecules have been shown to exhibit the same antigen-binding specificity as HLA-DRB1*0401 and to be functional in presenting antigens to T cells (29). The frequency of the HLA-DR allele in humans is 29% in Caucasian individuals, 10% in African American individuals, and 34% in other individuals (38), underscoring the translational value of the epitopes identified in these mice for humans. We recently demonstrated the feasibility of using the HLA-DR4 tg mouse for the identification of vaccine antigens against genital Chlamydia infection (39), demonstrating the value of the use of these animals in the rational selection of vaccine candidates.In this study, we utilized a robust biochemical membrane protein fractionation method, cytokine recall assays, and humanized HLA-DR4 tg mice to identify putative CD4⁺ T-cell-reactive antigens from U112. Moreover, using bioinformatics tools, we further validated one of the identified antigens (FTN_0119), designated Francisella outer membrane protein B (FopB), as a potential subunit vaccine candidate against pneumonic tularemia in HLA-DR4 tg mice.     

Potentiation of Excitotoxicity in Transgenic Mice Overexpressing Neuronal Cyclooxygenase-2

In this study we describe the generation of a transgenic mouse model with neuronal overexpression of the human cyclooxygenase-2, h(COX)-2, to explore its role in excitotoxicity. We report that overexpression of neuronal hCOX-2 potentiates the intensity and lethality of kainic acid excitotoxicity in coincidence with potentiation of expression of the immediate early genes c-fos and zif-268. In vitro studies extended the in vivo findings and revealed that glutamate excitotoxicity is potentiated in primary cortico-hippocampal neurons derived from hCOX-2 transgenic mice, possibly through potentiation of mitochondrial impairment. This study is the first to demonstrate a cause-effect relationship between neuronal COX-2 expression and excitotoxicity. This model system will allow the systematic examination of the role of COX-2 in mechanisms of neurodegeneration that involve excitatory amino acid pathways.     

严密型四环素调控系统转基因首建鼠的建立

目的制备ApoE-rtTA-tTS转基因小鼠1，为严密型四环素调控系统的体内研究提供调控部分的转基因小鼠，以便与反应部分小鼠交配得到双转基因小鼠。方法重组构建含目的基因的质粒pApoE-rtTA-tTS，应用显微注射法将其注入母鼠的受精卵，再植入代母输卵管，出生小鼠经PCR初步筛选出阳性，再经Southern杂交对阳性小鼠基因组DNA标本进一步鉴定。结果产生了2只整合ApoE-rtTA-tTS基因的首建鼠。结论成功制备了ApoE-rtTA-tTS转基因小鼠，为下一步建立严密型四环素调控系统的双转基因小鼠模型奠定了基础。     

Behavioral and Immune Changes in V-Ha-ras Transgenic Mice

Abstract

Transgenic mice (Oncomice) with an activated v-Ha-ras oncogene under the control of the mouse mammary tumor virus promoter develop mammary tumors. We wondered if the expression of the v-Ha-ras oncogene product would induce changes in mice behavioral activity, that could be associated with alterations in their immune system. Behavior was evaluated in an open field study considering line crossings and rears. Oncomice consistently showed less activity than FVB mice. Lieber-DeCarli diet decreased both types of activity in both strains. Cocaine treatment increased line crossings in both strains. Oncomice spleen and thymus cell supernatants contained higher levels of IL-2. Oncomice serum had higher levels of IL-1α. Our results suggest a direct association between higher levels of IL-1α and lower open field activity. Therefore, we can infer that the increased level of IL-1α found in Oncomice, could have a key role in oncogene induced immune and behavioral changes, and could be a requirement to facilitate its transforming activity.     

Increased Resistance to Taenia crassiceps Murine Cysticercosis in Qa-2 Transgenic Mice

We previously reported important differences in resistance to Taenia crassiceps murine cysticercosis between BALB/c substrains. It was suggested that resistance might correlate with expression of the nonclassic class I major histocompatibility complex (MHC) Qa-2 antigen; BALB/cAnN is Qa-2 negative and highly susceptible to T. crassiceps, whereas BALB/cJ expresses Qa-2 and is highly resistant. In this study, we investigated the role of Qa-2 in mediating resistance to cysticercosis by linkage analysis and infection of Qa-2 transgenic mice. In BALB/cAnN × (C57BL/6J × BALB/cAnN)F₁ and BALB/cAnN × (BALB/cJ × BALB/cAnN)F₁ backcrosses, the expression of Qa-2 antigen correlated with resistance to cysticercosis. Significantly fewer parasites were recovered from infected Qa-2 transgenic male and female mice than from nontransgenic mice of similar genetic background. These results clearly demonstrate that the Qa-2 MHC antigen is involved in resistance to T. crassiceps cysticercosis.

Taenia solium cysticercosis is a parasitic disease that seriously affects human health (24) and causes important economic losses in pig farming of developing countries (1) where conditions that favor parasite transmission persist. The essential role of pigs as an obligatory intermediate host in the parasite life cycle offers the opportunity to interfere with transmission by inducing acquired immunity through vaccination (10, 13, 18), by decreasing susceptibility through genetic manipulation (14), or both. Systematic exploration of the role of genetic factors in cysticercosis and the identification of protective immunogens are hampered by the high costs and slow data retrieval involved in studies with pigs. However, another cestode, Taenia crassiceps, that naturally infects rodents (3) is highly suitable for experimentation. It shows extensive antigenic cross-reactivity and cross-protective immunity with T. solium (7, 21); the antigenic similarity is such that T. crassiceps antigens can be used for immunodiagnosis of human cysticercosis (9). Furthermore, T. crassiceps and T. solium both have a typical two-host taeniid life cycle and morphologically and structurally related larval stages. Since T. crassiceps can reproduce asexually, experimental infection is readily attained by injecting the cysticerci in the peritoneal cavity of the mouse (3). Thus, T. crassiceps murine cysticercosis has been shown to be a useful experimental model of metacestode infection in the study of genetic factors involved in host resistance (2, 20) and underlying immunological mechanisms (19, 23, 25).Initial findings showed that genes linked to H-2 affect T. crassiceps growth in mice (20). Thus, significant differences in the extent of the parasitosis were found between mice carrying the H-2^d (BALB/cAnN and DBA/2) haplotype, which were the most susceptible, and mice with H-2^b (BALB/B, C57BL/6J, and C57BL/10J) or H-2^k (BALB/K, C3H/HeJ, and C3H/FeJ) haplotype, which were comparatively resistant. Further studies (2) showed low susceptibility of congenic and recombinant B10 mice, regardless of H-2 haplotype, indicating that genes in C57BL background confer resistance to the parasitosis such that they override the effect of H-2. The effect of genes outside H-2 on the control of parasite growth was also revealed by the differential susceptibility of three H-2^d BALB/c substrains, of which BALB/cAnN was highly susceptible, whereas BALB/cJ was highly resistant and BALB/cByJ displayed and intermediate degree of susceptibility (2). BALB/cAnN and BALB/cJ, which are genetically quite similar strains, differ in several phenotypes, including the expression of the Qa-2 antigen (11, 16). This antigen is a nonclassical class I major histocompatibility complex (MHC) molecule encoded by four genes (Q6 to Q9) located telomeric to the H-2D loci (11, 22). BALB/cJ (Qa-2^low), a Qa-2 expressor substrain, has only active Q6 and Q7 genes because Q8 and Q9 have fused, resulting in an inactive Q8/Q9^d gene (11, 15). In BALB/cAnN (Qa-2^null), an additional deletion of genomic DNA has occurred between the Q6 and Q7 genes, leading to their inactivation and accounting for the Qa-2 null expression (11). We proposed previously that differences in susceptibility to T. crassiceps observed between BALB/cJ and BALB/cAnN might be related to Qa-2 antigen expression (2). Here we describe that results of genetic linkage studies are entirely consistent with this hypothesis. Furthermore, a role of Qa-2 in mediating resistance to T. crassiceps was directly established by the diminution of parasite loads in infected Qa-2 transgenic mice.     

Transgenic and Knock-out Mice: Models of Neurological Disease

Besides providing useful model systems for basic science, studies based on modification of the mammalian germ line are changing our understanding of pathogenetic principles. In this article, we review the most popular techniques for generating specific germ line mutations in vivo and discuss the impact of various transgenic models on the study of neurodegenerative diseases.
The "gain of function" approach, i.e., ectopic expression of exogenous genes in neural structures, has deepened our understanding of neurodegeneration resulting from infection with papova viruses, picorna viruses, and human retroviruses. Further, inappropriate expression of mutated cellular molecules in the nervous system of transgenic mice is proving very useful for studying conditions whose pathogenesis is controversial, such as Alzheimer's disease and motor neuron diseases. As a complementary approach, ablation of entire cell lineages by tissue-specific expression of toxins has been useful in defining the role of specific cellular compartments. Modeling of recessive genetic diseases, such as Lesch-Nyhan syndrome, was helped by the development of techniques for targeted gene deletion (colloquially termed "gene knock-out"). Introduction of subtle homozygous mutations in the mouse genome was made possible by the latter approach. Such "loss of function" mutants have been used for clarifying the role of molecules thought to be involved in development and structural maintenance of the nervous system, such as the receptors for nerve growth factor and the P₀ protein of peripheral myelin. In addition, these models are showing their assets also in the study of enigmatic diseases such as spongiform encephalopathies.     

Metformin Decelerates Aging and Development of Mammary Tumors in HER-2/neu Transgenic Mice

Transgenic FVB/N female mice carrying HER-2/neu mammary cancer gene received metformin (1200 mg/liter) with drinking water 5 days a week starting from the age of 2 months until natural death. Metformin slightly reduced food consumption, but did not change water consumption and dynamics of weight gain. Mean life span of mice increased by 8% (p<0.05), in 10% long-living mice it was prolonged by 13.1%, and the maximum life span was prolonged by 1 month under the effect of metformin in comparison with the control. The rate of populational aging decreased by 2.26 times. The total incidence of mammary adenocarcinoma and their multiplicity did not change under the effect of metformin, while the latency of tumor development increased and the mean diameter of tumors decreased. Hence, we first demonstrated a geroprotective effect of metformin and its suppressive effect towards the development of mammary tumors.__________Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 6, pp. 691–694, June, 2005     

Transgenic and Knockout Mice in Research on Prion Diseases

Since the discovery of the prion protein (PrP) gene more than a decade ago, transgenetic investigations on the PrP gene have shaped the field of prion biology in an unprecedented way. Many questions regarding the role of PrP in susceptibility of an organism exposed to prions have been elucidated. For example mice with a targeted disruption of the PrP gene have allowed the demonstration that an organism that lacks PrP^c is resistant to infection by prions. Reconstitution of these mice with mutant PrP genes allowed investigations on the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Unexpectedly, transgenic mice expressing PrP with specific amino-proximal truncations spontaneously develop a neurologic syndrome presenting with ataxia and cerebellar lesions. A distinct spontaneous neurologic phenotype was observed in mice with internal deletions in PrP. Using ectopic expression of PrP in PrP knockout mice has turned out to be a valuable approach towards the identification of host cells that are capable of replicating prions. Transgenic mice have also contributed to our understanding of the molecular basis of the species barrier for prions. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of hemato- and lymphopoietic cells. Such studies have shed new light onto the mechanisms of prion spread and disease pathogenesis.     

Transgenic Expression of the Human MRP2 Transporter Reduces Cisplatin Accumulation and Nephrotoxicity in Mrp2-Null Mice

The chemotherapeutic drug cisplatin is actively transported into proximal tubules, leading to acute renal injury. Previous studies suggest that the multidrug resistance–associated protein 2 (Mrp2) transporter may efflux cisplatin conjugates from cells. We sought to determine whether the absence of Mrp2 alters the accumulation and toxicity of platinum in the kidneys of mice and whether transgenic expression of the human MRP2 gene could protect against cisplatin injury in vivo. Plasma, kidneys, and livers from vehicle- and cisplatin-treated wild-type and Mrp2-null mice were collected for quantification of platinum and toxicity. By 24 hours, twofold higher concentrations of platinum were detected in the kidneys and livers of Mrp2-null mice compared with wild types. Enhanced platinum concentrations in Mrp2-null mice were observed in DNA and cytosolic fractions of the kidneys. Four days after cisplatin treatment, more extensive proximal tubule injury was observed in Mrp2-null mice compared with wild-type mice. Kidneys from naive Mrp2-null mice had elevated glutathione S-transferase mRNA levels, which could increase the formation of cisplatin-glutathione conjugates that may be metabolized to toxic thiol intermediates. Transgenic expression of the human MRP2 gene in Mrp2-null mice reduced the accumulation and nephrotoxicity of cisplatin to levels observed in wild-type mice. These data suggest that deficiency in Mrp2 lowers platinum excretion and increases susceptibility to kidney injury, which can be rescued by the human MRP2 ortholog.Cisplatin is commonly used in chemotherapy regimens for the treatment of solid cancers. The success of cisplatin therapy is limited, in part, by kidney injury. Up to 37% of patients develop signs of nephrotoxicity after receiving a single dose of cisplatin despite strategies such as hydration to limit renal exposure.¹ This is problematic for patients because kidney injury can delay further treatment and limit the total number of chemotherapy cycles received, thereby reducing the overall efficacy of cisplatin-containing regimens.Previous studies have identified the contribution of uptake and efflux transporters to the renal secretion of cisplatin. The organic cation transporter 2 (Oct2; gene Slc22a2) is highly expressed in the kidneys and mediates the renal uptake of cisplatin.^2–5 Transgenic mice lacking Oct1 and Oct2 transporters exhibit decreased platinum (Pt) excretion and reduced nephrotoxicity.⁶ In addition, patients with cancer with a loss-of-function polymorphism in OCT2 (808G>T) are protected against cisplatin renal injury.^6,7 Additional data suggest that the copper transporter 1 (Ctr1; gene Slc31a1) participates in the renal uptake of cisplatin,^8–12 although recent work has found conflicting results.¹³ After uptake of cisplatin by Oct2 or Ctr1, the subsequent efflux of cisplatin into the renal lumen is mediated by the multidrug and toxin extrusion protein 1 (Mate1; gene Slc47a1).¹⁴ As expected, Mate1 knockout mice have increased renal concentrations of cisplatin and enhanced nephrotoxicity.¹⁴Once inside the renal cell, cisplatin is biotransformed by spontaneous hydrolysis to monoaquated and diaquated species. Formation of cisplatin aquated intermediates leads to electrophilic attack of cellular macromolecules, such as DNA and protein. The tripeptide glutathione (GSH) can also bind cisplatin and has been suggested to form a variety of nontoxic cisplatin conjugates, including monoplatinum-monoglutathione, diplatinum-monoglutathione, and bis-(glutathionato)-platinum.^15,16 It has been postulated that these conjugates have the potential for metabolism to reactive thiol intermediates. Previous studies suggest that the multidrug resistance–associated transporter 2 (Mrp2; gene Abcc2) can efflux GSH conjugates of cisplatin from cancer cells and confer resistance to cytotoxicity.^15,17,18 Overexpression of human (h)MRP2 increases cisplatin resistance by 10-fold in human embryonic kidney cells¹⁷ and reduces cisplatin accumulation by 30% in porcine kidney LLC-PK1 cells.¹⁸ Moreover, primary hepatocytes isolated from transport-deficient rats (Mrp2-deficient strain) exhibit increased binding of Pt to DNA and enhanced susceptibility to cisplatin cytotoxicity.¹⁹ However, the ability of Mrp2 to reduce the accumulation of cisplatin and/or its conjugates and limit renal injury in vivo is not well understood. Therefore, the purpose of this study was to determine whether loss of Mrp2 function enhances renal Pt accumulation and nephrotoxicity in mice and to evaluate the ability of the human MRP2 gene to rescue Mrp2-null mice from heightened cisplatin-induced nephrotoxicity in vivo.