RESUMO
In the lactating cow, mammary gland-specific hypomethylation occurs at two Hpa II sites in the 5'-flanking region of the alpha S1-casein gene, and one in the 3'-region. These sites, A, B and C, are at nucleotide position -1388, -774 and +18034, respectively, relative to the major transcription start site. Site B was hypomethylated when the alpha S1-casein gene was expressed, and methylated when not expressed. In transgenic mice containing the bovine alpha S1-casein 5' and 3' regulatory elements fused to the human lactoferrin (hLF) cDNA, in some cases similar methylation patterns of sites A and B, as compared to the situation in the cow, were observed. In five mouse lines (out of the seven analysed) expressing the transgene in the milk, site B was hypomethylated in the mammary gland, while it was methylated in liver. In the two other mouse lines, no correlation was found between transgene expression and mammary gland-specific hypomethylation of site B. One of the five mouse lines with transgene expression and showing mammary-gland-specific hypomethylation of site B was studied in detail. In this mouse line, induction of transgene expression preceded hypomethylation of site B.
Assuntos
Caseínas/genética , Caseínas/metabolismo , Metilação de DNA , DNA-Citosina Metilases/metabolismo , Glândulas Mamárias Animais/fisiologia , Animais , Sítios de Ligação , Bovinos , Feminino , Regulação da Expressão Gênica , Vetores Genéticos , Lactação , Camundongos , Camundongos Transgênicos , Mapeamento por Restrição , Transcrição GênicaRESUMO
The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine alpha S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 micrograms ml-1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.
Assuntos
Lactoferrina/biossíntese , Lactoferrina/genética , Glândulas Mamárias Animais/metabolismo , Leite/química , Proteínas Recombinantes de Fusão/biossíntese , Animais , Sequência de Bases , Caseínas/genética , Bovinos , Feminino , Regulação da Expressão Gênica/genética , Vetores Genéticos/genética , Humanos , Lactação/metabolismo , Masculino , Glândulas Mamárias Animais/crescimento & desenvolvimento , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/genética , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
We have combined gene transfer, by microinjection, with 'in vitro' embryo production technology, enabling us to carry out non-surgical transfer, to recipient cows, of microinjected embryos that have been cultured from immature oocytes. Using this approach, we have established 21 pregnancies from which 19 calves were born. Southern blot analysis proved that in two cases the microinjected DNA had been integrated in the host genome.
Assuntos
Animais Geneticamente Modificados , Caseínas/genética , Bovinos/genética , Lactoferrina/genética , Transfecção , Animais , Southern Blotting , Células Cultivadas , DNA/genética , DNA/isolamento & purificação , Transferência Embrionária , Desenvolvimento Embrionário e Fetal , Feminino , Humanos , Oócitos/fisiologia , Gravidez , Mapeamento por RestriçãoRESUMO
A human genomic fragment comprising the cellular retinoic acid binding protein (CRABP) gene was isolated. By using a panel of somatic cell hybrids, this gene could be assigned to human chromosome 15. Subsequently, a possible involvement of the CRABP gene in translocation (15;17) (q22;q11) positive acute promyelocytic leukemia (APL) was investigated. Although transposition of the CRABP gene could be demonstrated, we did not observe any gross CRABP rearrangement in a series of primary APL patients, nor in the acute myeloblastic leukemia cell line HL-60. Thus, the observed lack of CRABP expression in these leukemic cells may not be caused by disruption of its gene. CRABP maps to the region 15q22-qter.
Assuntos
Proteínas de Transporte/genética , Cromossomos Humanos Par 15 , Leucemia Promielocítica Aguda/genética , Proteínas de Neoplasias/genética , Northern Blotting , Southern Blotting , Cromossomos Humanos Par 17 , Clonagem Molecular , Humanos , Células Híbridas , Receptores do Ácido Retinoico , Translocação GenéticaRESUMO
The cellular retinoic acid binding protein is thought to be involved in the retinoic-acid-mediated signal transduction pathway. We have isolated the mouse cellular retinoic acid binding protein cDNA from an embryonal-carcinoma-derived cell line by using differential cDNA cloning strategies. In situ hybridization on sections of mouse embryos of various developmental stages indicated that the cellular retinoic acid binding protein gene, which we localized on mouse chromosome 9, is preferentially expressed in a subpopulation of neurectodermal cells. This restricted expression pattern suggests an important role for cellular retinoic acid binding protein in murine neurogenesis.
