RESUMEN
Nuclear transfer (NT) using transfected primary cells is an efficient approach for the generation of transgenic goats. However, reprogramming abnormalities associated with this process might result in compromised animals. We examined the health, reproductive performance, and milk production of four transgenic does derived from somatic cell NT. Goats were derived from two fetal cell lines, each transfected with a transgene expressing a different version of the MSP-1(42) malaria antigen, either glycosylated or non-glycosylated. Two female kids were produced per cell line. Health and growth of these NT animals were monitored and compared with four age-matched control does. There were no differences in birth and weaning weights between NT and control animals. The NT does were bred and produced a total of nine kids. The control does delivered five kids. The NT does expressing the glycosylated antigen lactated only briefly, probably as a result of over-expression of the MSP-1(42) protein. However, NT does expressing the non-glycosylated antigen had normal milk yields and produced the recombinant protein. These data demonstrated that the production of healthy transgenic founder goats by somatic cell NT is readily achievable and that these animals can be used successfully for the production of a candidate Malaria vaccine.
Asunto(s)
Clonación de Organismos , Cabras/fisiología , Proteína 1 de Superficie de Merozoito/genética , Técnicas de Transferencia Nuclear , Reproducción , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/fisiología , Conducta Animal , Estro , Glicosilación , Cabras/genética , Leche , Plasmodium/inmunologíaAsunto(s)
Antagonistas de los Receptores Histamínicos H1/toxicidad , Artropatías/inducido químicamente , Articulaciones/embriología , Anomalías Inducidas por Medicamentos/patología , Animales , Huesos/embriología , Huesos/patología , Fisura del Paladar/inducido químicamente , Fisura del Paladar/patología , Femenino , Artropatías/patología , Articulaciones/patología , Embarazo , RatasRESUMEN
We examined transgenic-cattle production by DNA microinjection into 1-, 2-, and 4-cell embryos, analyzing the impact on calf size and subsequent viability. Embryos were either collected at an abattoir by flushing oviducts from superovulated and artificially inseminated cows (in vivo-derived) or obtained by in vitro maturation and in vitro fertilization of oocytes aspirated from excised ovaries (in vitro-derived). A human serum albumin (hSA) milk-expression DNA construct was microinjected, either in one of the visible pronuclei of in vitro- and in vivo-derived 1-cell embryos or in the nuclei of two blastomeres of 2- and 4-cell in vivo-derived embryos. Microinjection-induced mortality (lysis and developmental block) was equivalent ( approximately 40%) for all microinjected embryos. Embryos were co-cultured with BRL cells in B-2 medium containing 10% fetal calf serum (FSC). Overall, embryo development to morulae/blastocysts was significantly greater for in vivo-derived ova (15.5%) than for in vitro-derived oocytes (9.3%). All morulae and blastocysts were transferred to synchronized recipient females on Days 6-8 post-fertilization. A total of 189 calves were delivered. Birth weights were significantly greater for calves generated from in vitro-derived oocytes compared with those generated from in vivo-derived oocytes. One transgenic bull calf was obtained from the microinjection of a 2-cell embryo. Fluorescence in situ hybridization (FISH) analysis of lymphocytes detected one transgenic integration site in all cells. Transmission frequency of the hSA transgene in embryos obtained through IVM/IVF/IVC utilizing the semen of the transgenic calf confirmed that it was not mosaic.
Asunto(s)
Peso al Nacer/genética , Transferencia de Embrión/métodos , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Razón de Masculinidad , Animales , Animales Modificados Genéticamente , Blastocisto/citología , Blastocisto/metabolismo , Blastómeros/citología , Blastómeros/metabolismo , Bovinos , ADN/administración & dosificación , ADN/genética , Embrión de Mamíferos/citología , Femenino , Fertilización In Vitro , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Hibridación Fluorescente in Situ , Masculino , Microinyecciones , Oocitos/citología , Oocitos/metabolismo , Reacción en Cadena de la Polimerasa , Recombinación Genética/genética , Semen/metabolismo , Procesos de Determinación del Sexo , Transgenes/genéticaAsunto(s)
Agonistas de los Receptores Histamínicos/farmacología , Antagonistas de los Receptores Histamínicos/farmacología , Receptores Histamínicos H2/efectos de los fármacos , Receptores Histamínicos H2/fisiología , Convulsiones/fisiopatología , Estimulación Acústica , Animales , Anticonvulsivantes/farmacología , Benzotiazoles , Convulsivantes/administración & dosificación , Dimaprit/administración & dosificación , Dimaprit/farmacología , Femenino , Masculino , Ratones , Ratones Endogámicos DBA , Pentilenotetrazol/administración & dosificación , Fenoxipropanolaminas , Piperidinas/farmacología , Convulsiones/etiología , Tiazoles/farmacologíaRESUMEN
In this study, we demonstrate the production of transgenic goats by nuclear transfer of fetal somatic cells. Donor karyoplasts were obtained from a primary fetal somatic cell line derived from a 40-day transgenic female fetus produced by artificial insemination of a nontransgenic adult female with semen from a transgenic male. Live offspring were produced with two nuclear transfer procedures. In one protocol, oocytes at the arrested metaphase II stage were enucleated, electrofused with donor somatic cells, and simultaneously activated. In the second protocol, activated in vivo oocytes were enucleated at the telophase II stage, electrofused with donor somatic cells, and simultaneously activated a second time to induce genome reactivation. Three healthy identical female offspring were born. Genotypic analyses confirmed that all cloned offspring were derived from the donor cell line. Analysis of the milk of one of the transgenic cloned animals showed high-level production of human antithrombin III, similar to the parental transgenic line.
Asunto(s)
Clonación de Organismos , Cabras/genética , Técnicas de Transferencia Nuclear , Animales , Animales Modificados Genéticamente , Antitrombina III/genética , Southern Blotting , Núcleo Celular/fisiología , Desarrollo Embrionario y Fetal , Femenino , Cabras/fisiología , Humanos , Hibridación Fluorescente in Situ , Masculino , Leche/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Embarazo , Proteínas Recombinantes/metabolismo , ReproducciónRESUMEN
Recombinant antibodies and their derivatives are increasingly being used as therapeutic agents. Clinical applications of antibodies often require large amounts of highly purified molecules, sometimes for multiple treatments. The development of very efficient expression systems is essential to the full exploitation of the antibody technology. Production of recombinant protein in the milk of transgenic dairy animals is currently being tested as an alternative to plasma fractionation for the manufacture of a number of blood factors (human antithrombin, human alpha-1-antitrypsin, human serum albumin, factor IX). The ability to routinely yield mg/ml levels of antibodies and the scale-up flexibility make transgenic production an attractive alternative to mammalian cell culture as a source of large quantities of biotherapeutics. The following review examines the potential of transgenic expression for the production of recombinant therapeutic antibodies.
Asunto(s)
Animales Modificados Genéticamente , Formación de Anticuerpos , Clonación Molecular/métodos , Leche/metabolismo , Animales , Femenino , Expresión Génica , Células Germinativas , Cabras , Humanos , Glándulas Mamarias Animales/metabolismo , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , TransgenesRESUMEN
Antibodies fused to human enzymes offer an alternative to specifically targeting tumors with antibodies linked to plant or bacterial toxins. Since large amounts of these reagents can be administered without eliciting non-specific toxicities, efficient methods of production are needed. The goal of this work was to express a complex immunoenzyme fusion protein (immunotoxin) in the mammary gland of transgenic mice. A chimeric mouse/human antibody directed against the human transferrin receptor (E6) was fused at its CH2 domain to the gene for a human angiogenic ribonuclease, angiogenin (Ang). It was expressed in the mammary gland of mice and secreted into mouse milk. Expression levels in milk were approximately 0.8 g/l. The chimeric protein retained antibody binding activity and protein synthesis inhibitory activity equivalent to that of free Ang. It was specifically cytotoxic to human tumor cells in vitro.
Asunto(s)
Inmunoglobulina G/biosíntesis , Glándulas Mamarias Animales/metabolismo , Proteínas de Neoplasias/biosíntesis , Receptores de Transferrina/inmunología , Ribonucleasa Pancreática/biosíntesis , Animales , Femenino , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ratones , Ratones Transgénicos , Leche , Proteínas de Neoplasias/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Ribonucleasa Pancreática/genética , Células Tumorales CultivadasAsunto(s)
Desarrollo Embrionario y Fetal/efectos de los fármacos , Antagonistas de los Receptores H2 de la Histamina/toxicidad , Animales , Cimetidina/farmacología , Cimetidina/toxicidad , Femenino , Crecimiento/efectos de los fármacos , Antagonistas de los Receptores H2 de la Histamina/farmacología , Embarazo , Ranitidina/farmacología , Ranitidina/toxicidad , Ratas , Reflejo/efectos de los fármacos , Reflejo de Sobresalto/efectos de los fármacosRESUMEN
Transgenic mice were produced by microinjection of a DNA construct composed of the bovine kappa-casein (kappa-CN) cDNA under the control of the goat beta-CN 5' promoter elements and 3' flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine kappa-CN RNA to the mammary gland and secretion of bovine kappa-CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine kappa-CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69%, and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine kappa-CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine kappa-CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did kappa-CN from bovine milk. A high degree of variation in the production of bovine kappa-CN within each of the transgenic lines was observed.
Asunto(s)
Caseínas/biosíntesis , Glándulas Mamarias Animales/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caseínas/genética , Bovinos , ADN/administración & dosificación , Femenino , Regulación del Desarrollo de la Expresión Génica , Cabras , Lactancia/metabolismo , Ratones , Ratones Transgénicos , Microinyecciones , Proteínas de la Leche/análisis , Proteínas de la Leche/biosíntesis , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Mapeo RestrictivoAsunto(s)
Conducta Animal/efectos de los fármacos , Desarrollo Óseo/efectos de los fármacos , Huesos/efectos de los fármacos , Antagonistas de los Receptores Histamínicos H1/farmacología , Efectos Tardíos de la Exposición Prenatal , Análisis de Varianza , Animales , Huesos/embriología , División Celular/efectos de los fármacos , Desarrollo Embrionario y Fetal/efectos de los fármacos , Femenino , Embarazo , RatasRESUMEN
Three transgenic females from a first generation transgenic male were induced to lactate between 11 and 12 months of age using a series of estrogen and progesterone injections. The milk contained human longer acting tissue plasminogen activator (LAtPA) at comparable concentrations (1-3 mg/ml) as occurred in the original founder female. In addition, the transgenic male was induced with a hormonal regime and was shown to produce 0.85 mg/ml of LAtPA. Milk protein gels indicated that the milk products (casein, IgG) were essentially normal. These experiments show that expression data for this vector can be evaluated in a shorter period of time in dairy goats than would be required through normal gestation and lactation schedules and can be used to identify the relative expression of transgenes in mammary tissue that would occur during normal lactation.
Asunto(s)
Animales Modificados Genéticamente , Caseínas/genética , Expresión Génica , Cabras , Lactancia/genética , Glándulas Mamarias Animales/metabolismo , Activador de Tejido Plasminógeno/genética , Animales , ADN Complementario/genética , Estradiol/farmacología , Femenino , Humanos , Masculino , Progesterona/farmacología , Proteínas Recombinantes de FusiónRESUMEN
Central histaminergic modulation of H1 rather than H2-receptors has been shown to modify epileptic activity. Compounds acting on the HIC- and H3-receptors were tested against chemically-induced seizures in mice. Compounds antagonising the microsomal and nuclear intracellular receptors (HIC) only modified seizures at doses where toxicity was observed. Antagonists of the histamine H3-receptor (thioperamide and burimamide) only potentiated the severity of clonic convulsions induced by picrotoxin, while impromidine (i.c.v.), an antagonist with H2-agonist activity, inhibited leptazol-induced seizures. The H3-agonist, (R)alpha-methylhistamine, potentiated chemically-induced seizures, but at lower doses there was slight inhibition.
Asunto(s)
Receptores Histamínicos H3/fisiología , Convulsiones/inducido químicamente , Animales , Anticonvulsivantes/farmacología , Conducta Animal/efectos de los fármacos , Convulsivantes/farmacología , Agonistas de los Receptores Histamínicos , Antagonistas de los Receptores Histamínicos , Antagonistas de los Receptores Histamínicos H1/farmacología , Masculino , Ratones , Ratones Mutantes Neurológicos , Pentilenotetrazol , PicrotoxinaRESUMEN
The construction and expression of a chimeric gene encoding a mouse/human antibody to the human transferrin receptor fused to the gene for angiogenin, a human homolog of pancreatic RNase, are described. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human angiogenin to a chimeric anti-transferrin receptor heavy chain gene. The antibody-enzyme fusion gene was introduced into a transfectoma that secretes the chimeric light chain of the same antibody, and cell lines were cloned that synthesize and secrete the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/ml. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis of K562 cells that express the human transferrin receptor but not toward a non-human-derived cell line that lacks this receptor. Whereas excess antibody to the same receptor did not itself inhibit protein synthesis, it was able to completely prevent the protein synthesis inhibition caused by the fusion protein. These results indicate that the cytotoxicity is due to a transferrin receptor-mediated mechanism involving the angiogenin portion of the fusion protein and demonstrate the feasibility of constructing recombinant antibody-RNase molecules capable of killing tumor cells bearing the transferrin receptor. The significance of the acquired cytotoxicity of a mouse/human chimeric antibody linked to a human protein may bear importantly in human therapeutic strategies that use mouse antibodies linked to toxins from plants or bacteria to target tumor cells. It is expected that the humanization of immunotoxins will lead to less toxicity and immunogenicity than currently available reagents.
Asunto(s)
Anticuerpos , Inmunotoxinas/toxicidad , Proteínas/genética , Receptores de Transferrina/genética , Ribonucleasa Pancreática , Animales , División Celular/efectos de los fármacos , Línea Celular , Humanos , Cinética , Leucemia Mielógena Crónica BCR-ABL Positiva , Ratones , Proteínas de Neoplasias/biosíntesis , Plásmidos , Proteínas/inmunología , Receptores de Transferrina/inmunología , Proteínas Recombinantes de Fusión/farmacología , Mapeo Restrictivo , TransfecciónRESUMEN
Here we describe the production of cystic fibrosis transmembrane conductance regulator (CFTR), the product of the gene associated with cystic fibrosis, in the milk of transgenic mice. Mammary specific expression was achieved by placing the CFTR cDNA under the control of the goat beta-casein gene promoter. By fractionation, CFTR was shown to be associated with the membranes that envelop milk fat globules as they are discharged from the apical surface of the mammary epithelia. Since milk fat globules may comprise up to 10% of whole milk, this represents a novel, inexpensive and efficient approach to produce CFTR and possibly other membrane-associated proteins. The availability of large quantities of CFTR could have important implications for the development of new therapies for cystic fibrosis.
Asunto(s)
Fibrosis Quística/genética , Proteínas de la Membrana/genética , Leche/fisiología , Animales , Caseínas/genética , Clonación Molecular , Regulador de Conductancia de Transmembrana de Fibrosis Quística , ADN/genética , ADN/aislamiento & purificación , Exones , Femenino , Humanos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/aislamiento & purificación , Ratones , Ratones Transgénicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Mapeo RestrictivoRESUMEN
Several toxins abolish cellular protein synthesis by attacking specific sites in 28 S RNA. One of these toxins, alpha-sarcin, is an RNase that also cleaves nonspecifically on the 3' side of purines in deproteinized RNA. Several other RNases were injected into Xenopus oocytes, examined for their ability to abolish protein synthesis, and compared with alpha-sarcin and ricin. Surprisingly, pancreatic RNase A or B abolished oocyte protein synthesis at concentrations (approximately 0.03 nM) comparable to, or lower than, the amount of alpha-sarcin (approximately 2 nM) or ricin (approximately 0.07 nM) required to abolish protein synthesis. RNases S and T1 only inhibited oocyte protein synthesis when used at concentrations approximately 10 x higher than RNase A whereas RNases C, T2, U2, and nuclease P1 required concentrations approximately 100 times higher than RNase A to abolish protein synthesis. There was a direct correlation between the degradation of oocyte RNA and the inhibition of protein synthesis. The RNase inhibitors RNasin and Inhibit-Ace injected into the oocyte both prevented RNase A from hydrolyzing oocyte rRNA and abolishing protein synthesis. Enzymatically inactive oxidized RNase A did not inhibit protein synthesis when injected into the oocyte. None of the RNases or alpha-sarcin abolished protein synthesis when added to oocyte extracellular medium. Angiogenin is a human plasma protein that induces blood vessel formation in chick embryos, has 35% amino acid identity with RNase A, and cleaves 18 S and 28 S RNA in rabbit reticulocyte lysates (St. Clair, D. K., Rybak, S. M., Riordan, J. F. & Vallee, B. L. (1988) Biochemistry 27, 7263-7268, and references therein). Recombinant angiogenin injected into oocytes abolished protein synthesis, and this toxic effect was inhibited by RNasin but was not inhibited by Inhibit-Ace. Unlike RNase A and the other nucleases that hydrolyzed cellular rRNA, no cleavage of 18 or 28 S RNA by recombinant angiogenin was seen at concentrations 100 x greater than necessary to abolish protein synthesis. Recombinant angiogenin must selectively attack specific RNA(s) or another target in the cell.
Asunto(s)
Endorribonucleasas , Inhibidores de la Síntesis de la Proteína/toxicidad , Ribonucleasa Pancreática , Ribonucleasas/toxicidad , Toxinas Biológicas/administración & dosificación , Animales , Toxina Diftérica/toxicidad , Proteínas Fúngicas/toxicidad , Microinyecciones , Oocitos , Proteínas/farmacología , ARN Ribosómico 28S/metabolismo , Ribonucleasas/administración & dosificación , Ribonucleasas/antagonistas & inhibidores , Ribonucleasas/química , Ricina/toxicidad , Xenopus laevisRESUMEN
The absorption of glycine and proline through the mid-intestines of chicks infected with Eimeria acervulina was impaired when the amino acids were presented to the mucosal surface as the dipeptide, glycylproline.
Asunto(s)
Pollos/parasitología , Coccidiosis/veterinaria , Dipéptidos/farmacocinética , Eimeria , Enfermedades de las Aves de Corral/metabolismo , Animales , Coccidiosis/metabolismo , Absorción Intestinal , MasculinoRESUMEN
The CD4 molecule is a high affinity receptor for the human immunodeficiency virus (HIV) envelope glycoprotein (gp160 or gp120). This glycoprotein is expressed on the surface membrane of cells infected with HIV. It has, therefore, been suggested that a soluble form of CD4 might be used as a targeting agent to deliver toxins selectively to cells infected with HIV. We demonstrate that CD4-Pseudomonas exotoxin A (PE) conjugates inhibit the proliferation of gp160-transfected Chinese hamster ovary cells and block HIV replication in virus-infected H9 cells. However, this inhibition of HIV replication appears to be incomplete since virus replication occurs following removal of the toxin conjugates from these cultures. Moreover, CD4-PE conjugates delay but do not inhibit HIV replication in human peripheral blood lymphocytes. These studies suggest that such conjugates should be assessed only as potential adjunctive therapies in the acquired immunodeficiency syndrome.