Proper Developmental Control of Human Globin Genes Reproduced by Transgenic Mice Containing a 160-kb BAC Carrying the Human β-Globin Locus

ABSTRACTFour independent bacterial artificial chromosome (BAC) clones containing the human B-globin gene locus were obtained from a human genomic BAC library. A 160-kb clone (186D7) carrying the entire human B-globin locus including the B-globin gene family, locus control region (LCR), and 3′ regulatory elements was used to transform mice. Four transgenic lines were generated by microinjecting the purified BAC DNA into the fertilized eggs. RNase protection analysis showed that the expression of human B-globin genes is tissue- and developmental stage-specific and the expression level is similar among the three independent transgenic lines which carry the entire human B-globin locus; however, no B-globin gene expression was detected in the transgenic mice lacking the LCR region. The results suggest that the transgenic mouse model system that we have produced and that uses BAC to study the complex human B-globin gene cluster is stable and reproducible. Our results also indicate that some newly characterized HSs upstream from the LCR appear not to play an important role in globin gene expression and switching, while the traditional LCR can ensure correct human B-globin gene expression in transgenic mice. The BAC-mediated transgenic system can be used for further studies to determine which kinds of cis-acting elements are included in regulating the developmental timing and the level of human B-globin gene expression.     

Human Granulocyte Colony-Stimulating Factor (G-CSF) Stimulates the In Vitro and In Vivo Development But Not Commitment of Primitive Multipotential Progenitors From Transgenic Mice Expressing the Human G-CSF Receptor

Granulocyte colony-stimulating factor (G-CSF) stimulates theproliferation and restricted differentiation of hematopoietic progenitors into neutrophils. To clarify the effects of G-CSF onhematopoietic progenitors, we generated transgenic (Tg) mice that hadubiquitous expression of the human G-CSF receptor (hG-CSFR). In clonalcultures of bone marrow and spleen cells obtained from these mice,hG-CSF supported the growth of myelocytic as well as megakaryocytic,mast cell, mixed, and blast cell colonies. Single-cell cultures oflineage-negative(Lin)c-Kit⁺Sca-1⁺ orSca-1 cells obtained from the Tg mice confirmed thedirect effects of hG-CSF on the proliferation and differentiation ofvarious progenitors. hG-CSF also had stimulatory effects on theformation of blast cell colonies in cultures using5-fluorouracil-resistant hematopoietic progenitors and clone-sortedLinc-Kit⁺Sca-1⁺ primitivehematopoietic cells. These colonies contained different progenitors inproportions similar to those obtained when mouse interleukin-3 was usedin place of hG-CSF. Administration of hG-CSF to Tg mice led tosignificant increases in spleen colony-forming and mixed/blast cellcolony-forming cells in bone marrow and spleen, but did not alter theproportion of myeloid progenitors in total clonogenic cells. Theseresults show that, when functional G-CSFR is present on the cellsurface, hG-CSF stimulates the development of primitive multipotentialprogenitors both in vitro and in vivo, but does not induce exclusivecommitment to the myeloid lineage.     

脂质体法转染人肿瘤坏死因子-α基因对裸鼠人肝癌移植瘤的抑制效应及其机制

背景：外源性肿瘤坏死因子（TNF）-α联合化疗药物对肿瘤的疗效较单独应用更佳,为肿瘤治疗提供了新的方向。目的：对裸鼠人肝癌移植瘤模型行脂质体介导的TNF-α基因瘤内转染,研究肝癌移植瘤的生长抑制情况及其机制。方法：经脂质体介导,以真核表达质粒pSVK3-TNF-α分别转染人肝癌细胞株SMMC-7721和裸鼠皮下SMMC-7721细胞移植瘤。测定SMMC-7721细胞的TNF-α浓度,甲基噻唑基四唑（MTT）法测定细胞杀伤率,流式细胞仪和原位末端标记（TUNEL）法检测细胞周期和凋亡情况。结果：TNF-α转基因治疗裸鼠人肝癌移植瘤结束第5d,移植瘤体积为（75.28±35.35）mm^3,显著低于对照组的（326.45±103.64）mm^3（P〈0.05）。TNF-α基因体外转染SMMC-7721细胞24、48、72h后,基因转染组每106个细胞的TNF-α表达量分别为（1680±187）pg、（1702±205）pg和（1650±164）pg,细胞杀伤率分别为（37.1±2.4）%、（79.4±4.3）%和（84.2±4.6）%。基因转染72h后,SMMC-7721细胞增殖指数为（30.5±3.2）%,显著低于对照组的（46.1±3.9）%（P〈0.05）;凋亡指数为（10.0±2.1）%,显著高于对照组的（2.7±0.4）%（P〈0.01）。结论：脂质体介导的TNF-α基因转染裸鼠人肝癌移植瘤可明显抑制肿瘤生长,其机制可能为影响肿瘤细胞生长周期以及诱导肿瘤细胞凋亡。     

Concentration of Fetal Red Blood Cells From a Mixture of Maternal and Fetal Blood by Anti-i Serum--An Aid to Prenatal Diagnosis of Hemoglobinopathies

Fetal red blood cells were concentratedfrom mixtures of maternal and fetal cells bydifferential agglutination with anti-i serum.This method will be useful for prenatal diagnosis of hemoglobinopathies when bloodobtained from the fetus is heavily contaminated by maternal cells. The method ispractical, except in very rare cases in whichthe maternal red cells are strongly agglutinated by anti-i.

Submitted on March 4, 1973 Revised on July 16, 1973 Accepted on July 29, 1973     

采用流式细胞仪检测艾滋病病毒1型膜蛋白小鼠免疫后IFN-γ的表达

目的通过流式细胞技术检测艾滋病病毒1型(HIV-1)Gp120膜蛋白免疫小鼠后的IFN-γ的表达。方法 构建表达HIV-1gp120基因的复制型重组痘苗病毒。与DNA疫苗联合免疫小鼠,用流式细胞仪检测小鼠脾淋巴细胞中IFN-γ的表达。结果 获得表达中国HIV-1流行株gp120基因的重组痘苗病毒rVVgp120、rVVM304,能正确表达Gp120蛋白。与DNA疫苗p120-VRC、pM304-VRC联合免疫小鼠后,用流式细胞仪检测到小鼠脾淋巴细胞能特异性的表达IFN-γ。结论 用流式细胞检测技术成功检测到免疫小鼠的脾淋巴细胞特异性的表达IFN-γ,用此方法可方便快捷地检测小鼠的细胞免疫效果。     

Induction of Brain Tumors by the Archetype Strain of Human Neurotropic JCPyV in a Transgenic Mouse Model

JC Virus (JCPyV), a member of the Polyomaviridiæ family, is a human neurotropic virus with world-wide distribution. JCPyV is the established opportunistic infectious agent of progressive multifocal leukoencephalopathy, a fatal demyelinating disease, which results from the cytolytic infection of oligodendrocytes. Mutations in the regulatory region of JCPyV determine the different viral strains. Mad-1 the strain associated with PML contains two 98 base pair repeats, whereas the archetype strain (CY), which is the transmissible form of JCPyV, contains only one 98 tandem with two insertions of 62 and 23 base pairs respectively. The oncogenicity of JCPyV has been suspected since direct inoculation into the brain of rodents and primates resulted in the development of brain tumors and has been attributed to the viral protein, T-Antigen. To further understand the oncogenicity of JCPyV, a transgenic mouse colony containing the early region of the archetype strain (CY), under the regulation of its own promoter was generated. These transgenic animals developed tumors of neural crest origin, including: primitive neuroectodermal tumors, medulloblastomas, adrenal neuroblastomas, pituitary tumors, malignant peripheral nerve sheath tumors, and glioblastomas. Neoplastic cells from all different phenotypes express T-Antigen. The close parallels between the tumors developed by these transgenic animals and human CNS tumors make this animal model an excellent tool for the study of viral oncogenesis.     

Identification of a Novel Human Rhinovirus C Type by Antibody Capture VIDISCA-454

Causative agents for more than 30 percent of respiratory infections remain unidentified, suggesting that unknown respiratory pathogens might be involved. In this study, antibody capture VIDISCA-454 (virus discovery cDNA-AFLP combined with Roche 454 high-throughput sequencing) resulted in the discovery of a novel type of rhinovirus C (RV-C). The virus has an RNA genome of at least 7054 nt and carries the characteristics of rhinovirus C species. The gene encoding viral protein 1, which is used for typing, has only 81% nucleotide sequence identity with the closest known RV-C type, and, therefore, the virus represents the first member of a novel type, named RV-C54.     

Adhesion-Dependent Release of Elastase From Human Neutrophils in a Novel, Flow-Based Model: Specificity of Different Chemotactic Agents

Neutrophils must adhere to the vessel wall, migrate, and degranulatein an ordered manner to perform their protective function. Disruptionof these processes may be pathogenic. Current knowledge of thedegranulation process is derived almost exclusively from studies onneutrophils in suspension, in which priming with the nonphysiologicalagent cytochalasin B is necessary to obtain elastase release inresponse to activating agents. To avoid this, we have adopted adifferent approach. Using a novel flow-based adhesion system, we havebeen able to quantify the release of elastase from the primary granulesof activated neutrophils adherent to immobilized platelets or purifiedreceptors without priming. Comparing stimuli, formyl tripeptide (fMLP),interleukin-8 (IL-8), activated complement fragment C5a, andplatelet-activating factor (PAF) all induced rapid conversion toCD11b/CD18 (MAC-1) -mediated stationary adhesion when perfused overneutrophils already rolling on platelet monolayers or purifiedP-selectin. However, fMLP, C5a, and IL-8, but not PAF, induced releaseof elastase from the adherent cells in minutes. Neutrophils stimulatedin suspension showed little degranulation. Treatment of neutrophilswith an inhibitor of 5-lipoxygenase-activating protein (MK886) andthus synthesis of leukotrienes (LTs) or with an antagonist of theLTB₄ receptor (LY223982) blocked the release of elastase.This indicated that endogenous synthesis of 5-lipoxygenase productssuch as LTs and autocrine activation of neutrophils was required forfMLP-driven elastase release. We hypothesize that the differentialability of PAF and fMLP to induce elastase release fromsurface-adherent neutrophils could arise from differential ability togenerate leukotrienes, such as LTB₄, and would be an appropriate mechanism for the control of elastase release during inflammation in vivo, where it is important that cytotoxic agents arenot released until activated neutrophils have migrated into theextravascular tissues.     

IL-13 Gene Translation is Arrested by a Novel Oligonucleotide in Cultured Human B-Lymphocytes

Antisense oligodeoxynucleotides (oligos) are the tools that bind to complementary sequence of targeted mRNA and block specifically protein translation. In the present study, a novel 20 mer oligo as an antisense for human IL-13 is introduced. This oligo is designed according to the IL-13 mRNA coding region and synthesized in two HPLC purified and FITC conjugated forms. Fluorescence oligo cell uptake is confirmed using flowcytometry and confocal microscopy, and cytotoxicity evaluation is performed using BrdU proliferation assay. Human tonsilar B-lymphocytes are purified by positive selection using magnetic cell sorting method and cultured with anti CD40 monoclonal antibody plus rIL-4 to induce IL-13 production. IL-13 antisense is added to medium and Real Time PCR for mRNA, and ELISA for protein assays. Data indicate that antisense application leads to down regulation and complete suppression of IL-13 protein with no significant effects on mRNA, suggesting in vitro protein translation arrest. Since Il-13 is a crucial cytokine in allergic conditions, we conclude that interference with the protein synthesis by a nontoxic and efficient antisense oligo can provide an available tool for the investigators on allergic diseases.     

Evidence That the Human Blood Group Antigens Gya and Hy Are Carried on a Novel Glycosylphosphatidylinositol-Linked Erythrocyte Membrane Glycoprotein

Immunoblotting under non-reducing conditions with purified human anti-Gya and anti-Hy locates both antigens to an erythrocyte membrane glycoprotein of apparent Mr 46,750-57,500. The antigens are destroyed on intact red cells by the enzymes pronase, trypsin and chymotrypsin, and by treatment with reducing agents. Immunoblotting with anti-Gya and anti-Hy to membranes prepared from red cells pre-treated with an Endo F preparation caused a mean reduction in apparent Mr of the glycoprotein by 11 kDa at the leading and trailing edges, when compared with control membranes. These results suggest that the glycoprotein has one or more complex N-glycans that are not completely sensitive to Endo F digestion on intact cells. The majority of Gya/Hy-active molecules are not tightly associated with the red cell membrane skeleton. A gross reduction in reactivity with anti-Gya and anti-Hy by immunoblotting was observed in red cell membranes from patients with paroxysmal nocturnal haemoglobinuria, suggesting a possible membrane linkage via glycosylphosphatidylinositol for the glycoprotein that carries the Gya and Hy antigens. Immunoprecipitation of the glycoprotein by anti-Gya showed that the protein migrates faster under reducing conditions (Mr 45,000-54,000). A putative dimer was also evident in the precipitates. The glycoprotein was demonstrated to be distinct from lymphocyte-function-associated antigen-3 (CD58), the LWab-active glycoprotein, the Fya-active glycoprotein, the Oka-active glycoprotein and the BRIC 125 glycoprotein (CD47).