RESUMO
A major obstacle to stem-cell gene therapy rests in the inability to deliver a gene into a therapeutically relevant fraction of stem cells. One way to circumvent this obstacle is to use selection. Vectors containing two linked genes serve as the basis for selection, with one gene encoding a selectable product and the other, a therapeutic protein. Applying selection in vivo has the potential to bring a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a 'cell-growth switch', allowing a minor population of genetically corrected cells into the therapeutic range. But strategies for achieving in vivo selection have traditionally relied on genes that confer resistance to cytotoxic drugs and are encumbered by toxicity. Here we describe a new system for in vivo selection that uses a 'cell-growth switch', allowing a minor population of genetically modified cells to be inducibly amplified, thereby averting the risks associated with cytotoxic drugs. This system provides a general platform for conditionally expanding genetically modified cell populations in vivo, and may have widespread applications in gene and cell therapy.
Assuntos
Separação Celular , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Neoplasias , Receptores de Citocinas , Animais , Plaquetas/citologia , Plaquetas/metabolismo , Southern Blotting , Transplante de Medula Óssea , Técnicas de Cultura de Células/métodos , Dimerização , Relação Dose-Resposta a Droga , Eritrócitos/citologia , Eritrócitos/metabolismo , Citometria de Fluxo , Granulócitos/citologia , Granulócitos/metabolismo , Proteínas de Fluorescência Verde , Cinética , Proteínas Luminescentes/metabolismo , Camundongos , Proteínas Oncogênicas/química , Proteínas Oncogênicas/metabolismo , Fenótipo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-kit , Receptores da Eritropoetina/química , Receptores da Eritropoetina/metabolismo , Receptores de Trombopoetina , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Fatores de Tempo , TransgenesRESUMO
Conspicuous differences in floral morphology are partly responsible for reproductive isolation between two sympatric species of monkeyflower because of their effect on visitation of the flowers by different pollinators. Mimulus lewisii flowers are visited primarily by bumblebees, whereas M. cardinalis flowers are visited mostly by hummingbirds. The genetic control of 12 morphological differences between the flowers of M. lewisii and M. cardinalis was explored in a large linkage mapping population of F2 plants n = 465 to provide an accurate estimate of the number and magnitude of effect of quantitative trait loci (QTLs) governing each character. Between one and six QTLs were identified for each trait. Most (9/12) traits appear to be controlled in part by at least one major QTL explaining >/=25% of the total phenotypic variance. This implies that either single genes of individually large effect or linked clusters of genes with a large cumulative effect can play a role in the evolution of reproductive isolation and speciation.
Assuntos
Plantas/genética , Característica Quantitativa Herdável , Mapeamento Cromossômico , Genes de Plantas , Fenótipo , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
Skeletal myoblast grafts can form contractile tissue to replace scar and repair injured myocardium. Although potentially therapeutic, generating reproducible and sufficiently large grafts remains a challenge. To control myoblast proliferation in situ, we created a chimeric receptor composed of a modified FK506-binding protein (F36V) fused with the fibroblast growth factor receptor-1 cytoplasmic domain. Mouse MM14 myoblasts were transfected with this construct and treated with AP20187, a dimeric F36V ligand, to induce receptor dimerization. Transfected myoblasts proliferated in response to dimerizer (comparable with basic fibroblast growth factor (bFGF) treatment), whereas the dimerizer had no effect on non-transfected cells. Similar to bFGF treatment, dimerizer treatment blocked myotube formation and myosin heavy chain expression and stimulated mitogen-activated protein (MAP) kinase phosphorylation in transfected cells. Non-transfected cells differentiated normally and showed no MAP kinase phosphorylation with dimerizer treatment. Furthermore, myoblasts treated with dimerizer for 30 days in culture reduced MAP kinase phosphorylation, withdrew from the cell cycle, and differentiated normally upon drug withdrawal, demonstrating reversibility of the effect. Thus, forced dimerization of the fibroblast growth factor receptor-1 cytoplasmic domain reproduces critical aspects of bFGF signaling in myoblasts. We hypothesize that in vivo administration of AP20187 following myoblast grafting may allow control over graft size and ultimately improve cardiac function.
Assuntos
Divisão Celular/fisiologia , Músculo Esquelético/citologia , Animais , Sequência de Bases , Células Cultivadas , Primers do DNA , Dimerização , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/fisiologia , Sistema de Sinalização das MAP Quinases , Camundongos , Ratos , TransfecçãoRESUMO
To determine whether cytokine-induced signals generate unique responses in multipotential hemopoietic progenitor cells, the signaling domains of 3 different growth factor receptors (Mpl, granulocyte-colony-stimulating factor [G-CSF] receptor, and Flt-3) were inserted into mouse primary bone marrow cells. To circumvent the activation of endogenous receptors, each signaling domain was incorporated into an FK506 binding protein (FKBP) fusion to allow for its specific activation using synthetic FKBP ligands. Each signaling domain supported the growth of Ba/F3 cells; however, only Mpl supported the sustained growth of transduced marrow cells, with a dramatic expansion of multipotential progenitors and megakaryocytes. These findings demonstrate that the self-renewal and differentiation of multipotential progenitor cells can be influenced through distinct, receptor-initiated signaling pathways.
Assuntos
Diferenciação Celular , Divisão Celular , Células-Tronco Hematopoéticas/citologia , Proteínas de Neoplasias , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Receptores de Citocinas , Receptores de Fator Estimulador de Colônias de Granulócitos/fisiologia , Animais , Sítios de Ligação , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Feminino , Fator Estimulador de Colônias de Granulócitos/farmacologia , Hematopoese , Megacariócitos/citologia , Proteínas de Membrana/farmacologia , Camundongos , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator Estimulador de Colônias de Granulócitos/química , Receptores de Fator Estimulador de Colônias de Granulócitos/genética , Receptores de Trombopoetina , Proteínas Recombinantes de Fusão , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/genética , Transfecção , Tirosina Quinase 3 Semelhante a fmsRESUMO
To investigate the potential for functional interactions between heterologous receptors, the cytoplasmic domains of 2 different receptors (c-Kit and Flt-3) were coexpressed in the interleukin-3-dependent cell line Ba/F3. The receptor signaling domains were presented in the context of fusion proteins, with c-Kit linked to the FK506 binding protein (FKBP12) and Flt-3 linked to the FRB domain of the FKBP12-rapamycin-associated protein. The fusions were brought into apposition with the use of chemical inducers of dimerization (CIDs). Two classes of CID were employed. FK1012 and its synthetic analogue AP1510 bring together 2 copies of the FKBP12 domain, thereby inducing homodimerization of the c-Kit(FKBP12) fusion. A second type of CID, rapamycin, brings together one FKBP12 domain and one FRB domain, resulting in heterodimerization of the c-Kit(FKBP12) and Flt-3(FRB) fusions. Ba/F3 cell growth was promoted not only by FK1012- or AP1510-induced homodimerization of the c-Kit(FKBP12) fusion (as reported previously), but also by rapamycin-induced c-Kit(FKBP12)-Flt-3(FRB) heterodimerization. These findings demonstrate the potential for a direct functional interaction between c-Kit and Flt-3. (Blood. 2001;97:3662-3664)
Assuntos
Divisão Celular , Proteínas Proto-Oncogênicas c-kit/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores Proteína Tirosina Quinases/fisiologia , Animais , Sítios de Ligação , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Dimerização , Interações Medicamentosas , Expressão Gênica , Interleucina-3/farmacologia , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-kit/genética , Receptores Proteína Tirosina Quinases/genética , Proteínas Recombinantes de Fusão , Transdução de Sinais , Sirolimo/metabolismo , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Proteínas de Ligação a Tacrolimo/metabolismo , Tirosina Quinase 3 Semelhante a fmsRESUMO
We have evaluated three DNA-based marker types for linkage map construction in Populus: RFLPs detected by Southern blot hybridization, STSs detected by a combination of PCR and RFLP analysis, and RAPDs. The mapping pedigree consists of three generations, with the F1 produced by interspecific hybridization between a P. trichocarpa female and a P. deltoides male. The F2 generation was made by inbreeding to the maximum degree permitted by the dioecious mating system of Populus. The applicability of STSs and RAPDs outside the mapping pedigree has been investigated, showing that these PCR-based marker systems are well-suited to breeding designs involving interspecific hybridization. A Populus genome map (343 markers) has been constructed from a combination of all three types. The length of the Populus genome is estimated to be 2400-2800 cM.
RESUMO
Cellular trafficking of growth factor receptors, including cross-talk among receptors at the cell surface, may be important for signal transduction in normal hematopoietic cells. To test this idea, the signaling domain of Mpl (the thrombopoietin receptor) was targeted to the plasma membrane, or to the cytoplasm of murine marrow cells, and the ability of the cells to proliferate and differentiate in response to Mpl dimerized at the plasma membrane or free in the cytoplasm was assessed. Constructs encoding the signaling domain of Mpl linked to an FK506 binding protein domain (to permit dimerization by the membrane-permeable ligand AP20187) with or without a myristylation sequence (to target the receptor to the plasma membrane) and a hemagglutinin epitope tag were generated and introduced into murine marrow cells using a murine stem cell virus (MSCV)-based retroviral vector. Both populations of transduced marrow cells proliferated in Iscoves modified Dulbecco medium-10% FCS-100 nM AP20187 without exogenous growth factors for more than 100 days and achieved greater than a 10(7)-fold expansion of cells by day 50 (n = 4 transductions). Growth was dimerizer dependent, and myeloid, erythroid, and megakaryocytic progenitors were generated. Activation of Mpl either at the plasma membrane or in the cytoplasm allowed for the terminal maturation of transduced progenitor cells. Introduction of membrane-targeted or cytoplasmic Mpl into fetal liver cells from homozygous JAK2 knock-out mice or wild-type littermates demonstrated that both forms of Mpl require JAK2 for signaling. These data show that the activation of Mpl independent of its normal plasma membrane location can support production of the full range of normal hematopoietic progenitor cells in vitro.