RESUMEN
Germline stem cells (GSCs) can be used for large animal transgenesis, in which GSCs that are genetically manipulated in vitro are transplanted into a recipient testis to generate donor-derived transgenic sperm. The objectives of this study were to explore a non-viral approach for transgene delivery into goat GSCs and to investigate the efficiency of nucleofection in producing transgenic sperm. Four recipient goats received fractionated irradiation at 8 weeks of age to deplete endogenous GSCs. Germ cell transplantations were performed 8-9 weeks post-irradiation. Donor cells were collected from testes of 9-week-old goats, enriched for GSCs by Staput velocity sedimentation, and transfected by nucleofection with a transgene construct harboring the human growth hormone gene under the control of the goat beta-casein promoter (GBC) and a chicken beta-globin insulator (CBGI) sequence upstream of the promoter. For each recipient, transfected cells from 10 nucleofection reactions were pooled, mixed with non-transfected cells to a total of 1.5 × 10(8) cells in 3 ml, and transplanted into one testis (n = 4 recipients) by ultrasound-guided cannulation of the rete testis. The second testis of each recipient was removed. Semen was collected, starting at 9 months after transplantation, for a period of over a year (a total of 62 ejaculates from four recipients). Nested genomic PCR for hGH and CBGI sequences demonstrated that 31.3% ± 12.6% of ejaculates were positive for both hGH and CBGI. This study provides proof-of-concept that non-viral transfection (nucleofection) of primary goat germ cells followed by germ cell transplantation results in transgene transmission to sperm in recipient goats.
Asunto(s)
Animales Modificados Genéticamente/genética , Células Germinativas/trasplante , Espermatozoides/fisiología , Trasplante de Células Madre/métodos , Transfección/métodos , Transgenes , Animales , Caseínas/genética , Pollos , Femenino , Genotipo , Células Germinativas/citología , Cabras , Hormona de Crecimiento Humana/genética , Hormona de Crecimiento Humana/metabolismo , Humanos , Inmunohistoquímica , Masculino , Regiones Promotoras Genéticas , Espermatozoides/citología , Células Madre/citología , Testículo/fisiología , Globinas beta/genéticaRESUMEN
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/genéticaRESUMEN
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
An individual found to be a true hermaphrodite at laparotomy, is presented. Cytogenetic studies which initially disclosed a 46,XX karyotype, conflicted with the anatomic presence of a testis. More extensive analysis of peripheral lymphocytes and skin fibroblasts revealed low level 46,XX/69,XXY mosaicism. DNA hybridization studies, using highly repeated Y chromosome specific probes, confirmed the rare presence of Y chromosome bearing cells. Such combined clinical and molecular studies can have an important impact on diagnosis and management of cases in which sex chromosome mosaicism is suspected.