RESUMO
To facilitate the screening for clones of transfected packaging cells producing a high yield of recombinant retrovirus, we present a fast and simple method for the isolation of overexpressing cells. By this method the efficiency of virus production can generally be enhanced 10- to 100-fold by application of high selection pressure. Cell lines which exhibit titers of up to 10(8) CFU/ml were obtained.
Assuntos
Clonagem Molecular/métodos , Retroviridae/crescimento & desenvolvimento , Retroviridae/genética , Células 3T3 , Animais , Linhagem Celular , Resistência Microbiana a Medicamentos/genética , Marcadores Genéticos , Vírus da Leucemia Murina/genética , Camundongos , Neomicina/farmacologia , Provírus/genética , Puromicina/farmacologia , Seleção Genética , Tetra-Hidrofolato Desidrogenase/genética , Cultura de Vírus , Replicação ViralRESUMO
The expression of certain genes has been reported to respond positively to sodium butyrate. This study demonstrates the same feature for two marker genes under the control of five different promoters. In all examples, the stimulatory effect is largest if one or especially two scaffold/matrix-attached regions (SAR/MAR elements) are present adjacent to the gene, and in one case, the stimulation depends entirely upon this situation. These results are observed with several SAR sequences including those obtained by oligomerizing short stretches of DNA surrounding a core motif. It is suggested that butyrate exerts important actions at the level of the chromatin structure.
Assuntos
Butiratos/farmacologia , Sequências Reguladoras de Ácido Nucleico/fisiologia , Transcrição Gênica/efeitos dos fármacos , Animais , Ácido Butírico , Cricetinae , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferon beta/biossíntese , Interferon beta/genética , Células L , Camundongos , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologiaRESUMO
Matrix attachment regions (MARs) are thought to separate chromatin into topologically constrained loop domains. A MAR located 5' of the human beta-interferon gene becomes stably base-unpaired under superhelical strain, as do the MARs flanking the immunoglobulin heavy chain gene enhancer; in both cases a nucleation site exists for DNA unwinding. Concatemerized oligonucleotides containing the unwinding nucleation site exhibited a strong affinity for the nuclear scaffold and augmented SV40 promoter activity in stable transformants. Mutated concatemerized oligonucleotides resisted unwinding, showed weak affinity for the nuclear scaffold, and did not enhance promoter activity. These results suggest that the DNA feature capable of relieving superhelical strain is important for MAR functions.
Assuntos
DNA/genética , Elementos Facilitadores Genéticos , Cadeias Pesadas de Imunoglobulinas/genética , Interferon beta/genética , Sequência de Bases , DNA/efeitos dos fármacos , DNA/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Humanos , Hidrazinas/farmacologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Matriz Nuclear/fisiologia , Oligodesoxirribonucleotídeos , Plasmídeos , Mapeamento por Restrição , Ésteres do Ácido Sulfúrico , Transcrição GênicaRESUMO
We have transfected DNA corresponding to the complete chromatin domain of human interferon beta (huIFN-beta) gene into mouse L cells. In this construct, which is flanked by scaffold-attached regions (SARs), the gene's transcription was enhanced 20-30-fold with respect to DNAs containing only the immediate regulatory elements. To elucidate the role of SAR elements in the transcriptional enhancement, their position was varied relative to several artificial promoter-gene combinations. It was found that SARs enhance general promoter functions in an orientation- and partially distance-independent manner; their effect is restricted to the integrated state of transfected templates. During the phase of transient expression, SAR elements were generally found to have an antagonizing effect.
