RESUMO
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.
Assuntos
Clonagem de Organismos , Cabras/fisiologia , Proteína 1 de Superfície de Merozoito/genética , Técnicas de Transferência Nuclear , Reprodução , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Comportamento Animal , Estro , Glicosilação , Cabras/genética , Leite , Plasmodium/imunologiaRESUMO
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.
Assuntos
Clonagem de Organismos , Cabras/genética , Técnicas de Transferência Nuclear , Animais , Animais Geneticamente Modificados , Antitrombina III/genética , Southern Blotting , Núcleo Celular/fisiologia , Desenvolvimento Embrionário e Fetal , Feminino , Cabras/fisiologia , Humanos , Hibridização in Situ Fluorescente , Masculino , Leite/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Gravidez , Proteínas Recombinantes/metabolismo , ReproduçãoRESUMO
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.
