RESUMO
UNLABELLED: ESSENTIALS: Anti-factor VIII (FVIII) inhibitory antibody formation is a severe complication in hemophilia A therapy. We genetically engineered and characterized a mouse model with complete deletion of the F8 coding region. F8(TKO) mice exhibit severe hemophilia, express no detectable F8 mRNA, and produce FVIII inhibitors. The defined background and lack of FVIII in F8(TKO) mice will aid in studying FVIII inhibitor formation. BACKGROUND: The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII (i.e. cross-reacting material, CRM) have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein. OBJECTIVES: We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8(TKO) strain) lacking the complete coding sequence of F8 and any FVIII CRM. METHODS: Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA. RESULTS: All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8(TKO) mice. The bleeding phenotype of F8(TKO) mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8(TKO) and E16 mice. CONCLUSIONS: We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice, which is valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background.
Assuntos
Fator VIII/genética , Deleção de Genes , Hemofilia A/genética , Hemostasia , Animais , Anticorpos/sangue , Testes de Coagulação Sanguínea , Compostos Cromogênicos , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Fator VIII/imunologia , Fator VIII/metabolismo , Fator VIII/farmacologia , Predisposição Genética para Doença , Hemofilia A/sangue , Hemofilia A/tratamento farmacológico , Hemofilia A/imunologia , Hemostasia/efeitos dos fármacos , Hemostasia/genética , Hemostáticos/imunologia , Hemostáticos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Índice de Gravidade de DoençaRESUMO
We briefly review the history of microRNA (miRNA) research and some of the lessons learnt. To provide some insights as to how and why miRNAs came into existence, we consider the evolution of the RNA interference machinery, miRNA genes, and their targets. We highlight the importance of systems biology approaches to integrate miRNAs as an essential subnetwork for modulating gene expression programs. Building accurate computational models that can simulate highly complex cell-specific gene expression patterns in mammals will lead to a better understanding of miRNAs and their targets in physiological and pathological situations. The impact of miRNAs on medicine, either as potential disease predisposing factors, biomarkers, or therapeutics, is highly anticipated and has started to reveal itself.
Assuntos
Genes , MicroRNAs/genética , MicroRNAs/metabolismo , Animais , Expressão Gênica , Genoma , Mamíferos/genética , Mamíferos/metabolismo , Interferência de RNARESUMO
The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.
Assuntos
Sítios de Ligação Microbiológicos/genética , Bacteriófago P1/enzimologia , Bacteriófago P1/genética , Integrases/metabolismo , Mutagênese Sítio-Dirigida/genética , Recombinação Genética/genética , Proteínas Virais/metabolismo , Sequência de Bases , Western Blotting , Dimerização , Escherichia coli/genética , Genes Reporter/genética , Genes Virais/genética , Integrases/genética , Sequências Repetitivas de Ácido Nucleico/genética , Deleção de Sequência/genética , Proteínas Virais/genéticaRESUMO
Pit cells are liver-specific natural killer (NK) cells and belong to the group of sinusoidal cells, together with Kupffer, endothelial, and fat-storing cells. Pit cells are lymphoid cells containing specific granules, classifying them also as large granular lymphocytes (LGL). They probably originate from the bone marrow, circulate in the blood, and marginate in the liver, where they develop into pit cells by lowering their density and increasing the number of granules, which decrease in size. Pit cells remain in the liver about 2 weeks and are dependent on Kupffer cells. Pit cells also proliferate locally, when stimulated with interleukin-2, biological response modifiers, or other agents. Pit cells adhere to tumor target cells during killing. They possess a high level of natural cytotoxicity against a variety of tumor cell lines, which is comparable to the cytotoxicity level of lymphokine-activated killer (LAK) cells. Tumor cell killing is synergistically enhanced when pit cells attack tumor cells together with Kupffer cells. Further investigations are needed to clarify the mechanisms of pit cell cytotoxicity and the role of these cells in killing virus-infected cells, such as during viral hepatitis.
