RESUMEN
Transgenic mice expressing human urokinase, as well as animals expressing human urokinase receptor under the control of the murine mammary tumor virus (MMTV) long terminal repeat, were established. In the vast majority of the founder animals and their descendants, the transgene was completely methylated, corresponding to down-regulation of transgene expression in the mammary gland. Two lineages with human urokinase receptor as the transgene with mixed methylation of the transgenes were analyzed in more detail. We show here for the first time that the methylation status of the transgene is identical in different organs of an animal, but may differ from animal to animal among the descendents. In the mammary gland, complete methylation of the transgene was incompatible with expression; unmethylated and mixed methylation transgenes gave rise to expression at the RNA as well as at the protein level. The methylation observed was not the consequence of an imprinting process. Surprisingly, in organs other than the mammary gland, such as liver, kidney and spleen, weak expression of the transgene was noted independent of the methylation status of the MMTV promoter. With respect to the molecular mechanism it is unresolved whether the human growth hormone sequence of the transgene harbors a methylation inducing element responsible for the observed methylation pattern.
Asunto(s)
Virus del Tumor Mamario del Ratón/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Transgenes , Activador de Plasminógeno de Tipo Uroquinasa/genética , Células 3T3 , Animales , Metilación de ADN , Precursores Enzimáticos/genética , Vectores Genéticos , Humanos , Ratones , Microinyecciones , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Mapeo Restrictivo , TransfecciónRESUMEN
Chimerization of antibodies (Ab) by cloning the V (variable) regions encoding the light and heavy chains with degenerate oligodeoxyribonucleotide primers matching to framework region 1 and to the joining regions, leads to Ab with altered amino acids at the N-terminus compared to those of the parental Ab. This is due to N-terminal framework 1 sequences in the expression vectors [Larrick et al., Bio/Technology 7 (1989) 937-938; Le Boeuf et al., Gene (1989) 371-377; Orlandi et al., Proc. Natl. Acad. Sci. USA 86 (1989) 3833-3837]. This might lead to Ab with altered affinity to the antigen due to interaction of framework sequences with complementarity determining regions. Moreover, some V regions may be refractory to cloning by this procedure. Here, we describe a method to circumvent these potential problems. The V regions for both chains of the Ab are cloned by inverse polymerase chain reaction (PCR) with primers matching the known constant region sequences of the Ab. After sequencing, PCR fragments corresponding to the V regions of both chains are inserted in-frame into appropriate expression vectors leading to Ab with unaltered N-terminal sequences after expression in mammalian cells. The procedure is illustrated with an Ab directed against the beta chain of the human interleukin-2 receptor.
Asunto(s)
Anticuerpos Monoclonales/genética , Clonación Molecular/métodos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes de Fusión/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , ADN , Vectores Genéticos , Humanos , Región Variable de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , ARN Mensajero/genética , Receptores de Interleucina-2/genética , Receptores de Interleucina-2/inmunología , Proteínas Recombinantes de Fusión/inmunología , Células Tumorales CultivadasRESUMEN
50 cases, in which phlebography was indicated by clinical symptoms of deep venous thrombosis, were examined by duplex-sonography. In comparison with the phlebographic findings, duplex-sonography was 100% sensitive in detecting thrombosis of the popliteal, femoral and iliac veins and 89% respectively 95% and 100% specific. A sonographic performance of the calf veins was only possible in 44%, the sensitivity in these cases was 92%, specificity 50%. Thus, the non-invasive duplex-sonography is a very appropriate method to diagnose thrombosis of the popliteal region and proximal. For proving or excluding thrombosis of the calf veins phlebography remains necessary.