Generation of Pax2-Cre mice by modification of a Pax2 bacterial artificial chromosome

We generated a transgenic mouse line named E1-Ngn2/Cre that expresses Cre recombinase and GFP under the control of the E1 enhancer element of the gene Ngn2 (Scardigli et al.: Neuron 31:203-217, 2001). Cre-recombinase activity and GFP fluorescence are consistent with the reported expression pattern controlled by the E1-Ngn2 enhancer. Recombination was detected in the progenitor domains p1 and p2 in the ventricular zone of the neural tube and in distinct domains of the pretectum, the dorsal and ventral thalamus, the tegmentum of the mesencephalon, and the hindbrain. In the developing cortex, Cre-recombinase activity is confined to a subpopulation of progenitors predominantly in the region of the ventral and lateral pallium. The E1-Ngn2/Cre mouse line thus provides an excellent novel tool for a region-specific conditional mutagenesis in the developing CNS.     

Homologous recombination as an evolutionary force in the avian influenza A virus

Avian influenza A viruses (AIVs), including the H5N1, H9N2,and H7N7 subtypes, have been directly transmitted to humans,raising concerns over the possibility of a new influenza pandemic.To prevent a future avian influenza pandemic, it is very importantto fully understand the molecular basis driving the change inAIV virulence and host tropism. Although virulent variants ofother viruses have been generated by homologous recombination,the occurrence of homologous recombination within AIV segmentsis controversial and far from proven. This study reports threecirculating H9N2 AIVs with similar mosaic PA genes descendedfrom H9N2 and H5N1. Additionally, many homologous recombinantsare also found deposited in GenBank. Recombination events canoccur in PB2, PB1, PA, HA, and NP segments and between lineagesof the same/different serotype. These results collectively demonstratethat intragenic recombination plays a role in driving the evolutionof AIVs, potentially resulting in effects on AIV virulence andhost tropism changes.     

Homologous recombination: a basis for targeted genome optimization in crop species such as maize

In an attempt to understand the feasibility of future targeted genome optimization in agronomic crops, we tested the efficiency of homologous recombination-mediated sequence insertion upon induction of a targeted DNA double-strand break at the desired integration site in maize. By the development of an efficient tissue culture protocol, and with the use of an I- Sce I gene optimized for expression in maize, large numbers of precisely engineered maize events were produced in which DNA integration occurred very accurately. In a subset of events examined in detail, no additional deletions and/or insertions of short filler DNA at the integration site were observed. In 30%–40% of the recovered events, no traces of random insertions were observed. This was true for DNA delivery by both Agrobacterium and particle bombardment. These data suggest that targeted double-strand break-induced homologous recombination is a superior method to generate specific desired changes in the maize genome, and suggest targeted genome optimization of agronomic crops to be feasible.     

Oncogenicity of virulent Marek's disease virus cloned as bacterial artificial chromosomes

Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that induces T-cell lymphomas in poultry. We report the construction of bacterial artificial chromosome (BAC) clones of the highly oncogenic RB-1B strain by inserting mini-F vector sequences into the U(S)2 locus. MDV reconstituted from two BAC clones induced rapid-onset lymphomas similar to those induced by the wild-type virus. Virus reconstituted from another BAC clone that showed a 7.7-kbp deletion in the internal and terminal unique long repeat regions was nononcogenic, suggesting that the deleted region may be associated with oncogenicity. The generation of the oncogenic BAC clones of MDV is a significant step in unraveling the oncogenic determinants of this virus.     

中国口蹄疫病毒同源重组研究

从GenBank数据库中获得在我国分离的16株口蹄疫病毒全基因组序列,进而运用常规的系统发生方法分析了这16株病毒的同源重组情况,发现5株重组毒株．这些重组病毒主要来源于亚洲Ⅰ型(Asia1)和O型病毒间的重组．这些重组事件的鉴定也表明口蹄疫病毒间的交叉感染在我国比较常见．另外,在我国还出现了由于Asia1型和O型病毒重组后导致病毒血清型发生转化的现象．这些结果解释了我国口蹄疫病毒(FMDV)遗传多样性和抗原多变性的成因,提示了我国在口蹄疫预防、治疗方面所面临的复杂局面．     

EB病毒及其疫苗的研究进展

EB病毒广泛存在于人群中,它的潜伏感染与多种疾病密切相关,包括鼻咽癌等恶性肿瘤,研制疫苗用于EB病毒相关疾病的预防和治疗十分必要。本综述了EB病毒的结构及基因表达特点、EB病毒潜伏期感染基因表达及潜伏期基因产物的功能,以及研制EB病毒疫苗的靶抗原的选择。     

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.     

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis.Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions.The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program.In this short review we touch upon aspects which are the subject of our present work.We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells.The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.     

Induction of the Epstein-Barr virus latent membrane protein 2 antigen-specific cytotoxic T lymphocytes using human leukocyte antigen tetramer-based artificial antigen-presenting cells

Cytotoxic T lymphocytes (CTLs) specific for the Epstein-Barr virus (EBV) latent membraneprotein 2 (LMP2) antigen are important reagents for the treatment of some EBV-associated malignancies,such as EBV-positive Hodgkin's disease and nasopharyngeal carcinoma.However,the therapeutic amount ofCTLs is often hampered by the limited supply of antigen-presenting cells.To address this issue,an artificialantigen-presenting cell (aAPC) was made by coating a human leukocyte antigen (HLA)-pLMP2 tetramericcomplex,anti-CD28 antibody and CD54 molecule to a cell-sized latex bead,which provided the dual signalsrequired for T cell activation.By co-culture of the HLA-A2-LMP2 bearing aAPC and peripheral bloodmononuclear cells from HLA-A2 positive healthy donors,LMP2 antigen-specific CTLs were induced andexpanded in vitro.The specificity of the aAPC-induced CTLs was demonstrated by both HLA-A2-LMP2tetramer staining and cytotoxicity against HLA-A2-LMP2 bearing T2 cell,the cytotoxicity was inhibited bythe anti-HLA class Ⅰ antibody (W6/32).These results showed that LMP2 antigen-specific CTLs could beinduced and expanded in vitro by the HLA-A2-LMP2-bearing aAPC.Thus,aAPCs coated with an HLA-pLMP2 complex,anti-CD28 and CD54 might be promising tools for the enrichment of LMP2-specificCTLs for adoptive immunotherapy.