RESUMO
The use of live bioreactors for the expression of human genes in the mammary gland of transgenic animals is one of the most cost-effective ways for the production of valuable recombinant therapeutic proteins. Among the transgenic species used so far, rabbits are good candidates for the expression of tens to hundreds of grams of complex proteins in the milk during lactation. The lactating mammary gland of rabbits has proven to be effective in the processing of complex proteins. In this work. the potential use of rabbits as bioreactors is discussed based on our results and the published data.
Assuntos
Produtos Biológicos/biossíntese , Biotecnologia/métodos , Proteínas do Leite/biossíntese , Coelhos/genética , Proteínas Recombinantes de Fusão/biossíntese , Animais , Animais Geneticamente Modificados , Produtos Biológicos/genética , Caseínas/genética , Feminino , Previsões , Regulação da Expressão Gênica , Genes Sintéticos , Vetores Genéticos , Humanos , Masculino , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Leite/genética , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/genética , Transgenes , Proteínas do Soro do LeiteRESUMO
The expression of chimeric genes in the mammary gland of transgenic farm animals has become an alternative for the large-scale production of recombinant proteins and for the modification of milk composition. In this paper, we show that a mouse/human chimeric antibody against the human CD6 leukocyte antigen can be assembled and correctly folded by the mammary gland, and secreted to milk, where it maintains its specificity. The base sequences encoding for the heavy and light chain variable regions of the anti-CD6 mouse monoclonal antibody IOR-T1 were cloned by the polymerase chain reaction from hybridoma cDNA, coupled to human heavy and light chain constant region genes, and inserted in a vector containing the 5' regulatory region of the rabbit whey acidic protein gene. Transgenic mice were produced by conventional pronuclei microinjection techniques. Integration and transgene copy number were determined by Southern blot. Assembled human immunoglobulin was detected in milk using a sandwich ELISA. Expression levels of chimeric antibodies in milk were determined to be around 400 micrograms/ml by Western blot, using CHO-derived chimeric IOR-T1 antibodies as reference. The chimeric antibodies produced in milk recognized human peripheral blood T lymphocytes by indirect immunofluorescence, with the classical patch-like pattern of IOR-T1.
Assuntos
Anticorpos/genética , Antígenos CD/imunologia , Antígenos de Diferenciação de Linfócitos T/imunologia , Leite/química , Proteínas Recombinantes de Fusão/biossíntese , Animais , Western Blotting , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Glândulas Mamárias Animais/metabolismo , Camundongos , Camundongos Transgênicos , CoelhosRESUMO
Gene farming is one of the most promising areas in modern biotechnology. To assay the potential usefulness of transgenic rabbits as bioreactors, one call embryos were microinjected with a chimeric gene comprising 5' sequences from mouse whey acidic protein gene (mWAP) linked to the human growth hormone (hGH) gene. Transgenic animals were obtained and the presence of the foreign protein was detected in the milk and serum of these animals at levels of up to 50 micrograms ml-1 and 0.6 ng ml-1, respectively. Founder transgenics were able to transmit the microinjected gene to the first filial generation in a Mendelian fashion. These results showed that transgenic rabbits could constitute a suitable system for the rapid production of recombinant proteins in the milk of lactating females.
Assuntos
Hormônio do Crescimento/biossíntese , Animais , Animais Geneticamente Modificados , Sequência de Bases , Biotecnologia , Quimera/genética , Clonagem Molecular , Primers do DNA/genética , Escherichia coli/genética , Feminino , Expressão Gênica , Vetores Genéticos , Hormônio do Crescimento/sangue , Hormônio do Crescimento/genética , Humanos , Lactação , Masculino , Leite/metabolismo , Proteínas do Leite/genética , Dados de Sequência Molecular , Plasmídeos/genética , Coelhos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/sangue , Proteínas Recombinantes/genéticaRESUMO
One-cell embryos from mice and rabbits were microinjected with a hybrid gene composed of 1.6 kilobases (kb) promoter/regulatory sequences of the bovine alphaS1 casein gene fused to the complementary DNA (cDNA) encoding for the human tissue plasminogen activator (htPA) and 3'untranslated sequences from rabbit beta-globin and SV 40 genes. Transgenic mice and rabbits that carry the htPA gene were obtained. In mice, 11 founder females were generated, and 6 of them expressed low levels (about 50 mug/ml) of htPA in their milk. Some of the transgenic mice showed rearrangements of the microinjected DNA sequences as judged by Southern blot analysis. A position-dependent expression of the transgene is suspected to occur. The only live-born founder transgenic rabbit obtained was a male, and it transmitted the transgene in a Mendelian fashion to F1 females, which expressed htPA at very low levels (8 to 50 ng/ml). Although the 1.6-kb bovine alphaS1 casein promoter that was used directs the synthesis of htPA specifically to the mammary gland, it may not be sufficient for a high level of expression.
