RESUMEN
The skin cells of newborn mice were stably transformed in vivo with the aid of electroporation. The plasmid DNA was introduced subcutaneously followed by high-voltage pulses applied to the skin pleat. NEO-resistant colonies were found in primary cell cultures obtained from the treated skin. The experiments show that in vivo electroporation can be used for the introduction of plasmid DNA into skin cells of mouse.
Asunto(s)
ADN/genética , Plásmidos , Piel/citología , Animales , Animales Recién Nacidos , Northern Blotting , Southern Blotting , Línea Celular Transformada , Electricidad , Fibroblastos/citología , Ratones , Ratones Endogámicos CBA , Hibridación de Ácido Nucleico , ARN/análisis , TransfecciónRESUMEN
NIH 3T3 mouse cells were transfected by the plasmid pSV3neo (G418-resistant) with the help of high-velocity mechanical DNA injection based on the principle of bombarding cells with tungsten particles covered with the DNA. Stable transformants were obtained. Dot-hybridization and Southern analysis revealed the integration into the genome of 5-20 copies per cell of original plasmid DNA. The plasmid DNA was shown to have tandem organization.
Asunto(s)
ADN/administración & dosificación , Transfección/métodos , Transformación Genética , Animales , Línea Celular , ADN/genética , Desoxirribonucleasa EcoRI , Ratones , Microinyecciones , Hibridación de Ácido Nucleico , Plásmidos , TungstenoRESUMEN
Mouse and rat liver, kidney and mammary gland explants were bombarded with high-velocity microprojectiles carrying a chloramphenicolacetyl transferase gene under different promoters (pTAT-cat, p chi-Casein-cat, p beta-Casein-cat). The expression of a CAT gene was revealed in all organ explants 24 h after transfection. The most pronounced expression was found when a TAT-CAT construction was used. In experiments in vivo rat liver was bombarded in situ with microprojectiles carrying pTAT-cat DNA. A marked activity of the CAT gene was detected 24 h after the bombardment.
Asunto(s)
Cloranfenicol O-Acetiltransferasa/genética , Riñón/enzimología , Hígado/enzimología , Glándulas Mamarias Animales/enzimología , Animales , ADN/química , Técnicas In Vitro , Regiones Promotoras Genéticas , Ratas , Ratas Endogámicas , Transfección , Transformación GenéticaRESUMEN
New methods of transfection of mammalian cells are discussed in the review. The principles of each method and frequency of genetic transformation are pointed out as well as their perspectives. The retrovirus constructions are noteworthy, because of the progress attained in their creation.
Asunto(s)
ADN/genética , Transfección , Animales , Métodos , Retroviridae/genética , Transformación GenéticaRESUMEN
Introduction of the plasmid containing the methotrexate-resistant (Mtx-r) bacterial gene of dihydrofolate reductase (DHFR) under the control of the early promoter of SV 40 into the donor bone cells of the mouse with subsequent transplantation of the cells into lethally irradiated mice results in the increase in the life span of mice under conditions of methotrexate selection. It is due to the stable transformation of the bone marrow colony-forming cells with the plasmic DNA and the synthesis of the bacterial Mtx-r DHFR in the spleen and bone marrow of the recipient mouse.
Asunto(s)
Células de la Médula Ósea , Genes Bacterianos , Células Madre Hematopoyéticas/citología , Tetrahidrofolato Deshidrogenasa/genética , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/enzimología , ADN/genética , Farmacorresistencia Microbiana , Antagonistas del Ácido Fólico , Regulación de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/efectos de los fármacos , Técnicas Genéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/enzimología , Masculino , Metotrexato/antagonistas & inhibidores , Ratones , Ratones Endogámicos CBA , Plásmidos , Regiones Promotoras Genéticas , Virus 40 de los Simios/genética , Bazo/citología , Bazo/efectos de los fármacos , Bazo/enzimología , Irradiación Corporal TotalRESUMEN
In mice obtained after microinjection into the male pronucleus of fertilized eggs of the plasmid, containing the bacterial gene of dihydrofolate reductase (DHFR), under the control of the early promotor of the simian virus 40 (SV40), an integration of the foreign DNA into the mouse genome is found. About 30% of the treated animals contain the integrated plasmid DNA sequences, i.e. are transgenic. In 2 of 7 mice, containing the introduced plasmid in their genome, the methotrexate-resistant DHFR activity is found in the kidney and spleen, which may be due to the expression of gene DHFR. The plasmid DNA sequences and the ability to synthesise the methotrexate-resistant enzyme DHFR are transmitted to the next generation of mice.
Asunto(s)
Genes Bacterianos , Tetrahidrofolato Deshidrogenasa/genética , Transformación Genética , Animales , ADN Bacteriano/genética , Femenino , Antagonistas del Ácido Fólico , Regulación de la Expresión Génica , Masculino , Metotrexato/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Hibridación de Ácido Nucleico , Plásmidos , Regiones Promotoras Genéticas , Virus 40 de los Simios/genéticaRESUMEN
A plasmid containing the bacterial gene of methotrexate-resistant dihydrofolate reductase (dhfr), under the control of early SV40 promoter, was introduced into murine teratocarcinoma CBA9H6 cells. From the whole pool of teratocarcinoma cells, which survived after transient methotrexate selection in vivo, the individual cells were isolated to give rise to 15 clones of tumors. Six of the 15 clones displayed nucleotide sequences of the original vector containing pBR322 sequences and the early SV40 promoter region; however, the bacterial dhfr gene was absent from the transformant clones. Possible causes of the loss of introduced dhfr gene from teratocarcinoma cells under non-selective conditions are discussed.