Reproductive parameters and the use of MOET in transgenic founder goat carrying the human granulocyte colony-stimulating factor (hG-CSF) gene

This study aimed to monitor estrous cycle parameters of a human granulocyte colony-stimulating factor (hG-CSF)-transgenic founder female goat and to perform superovulation and embryo recovery (surgical or transcervical method) for further transfer to recipients to quickly obtain offspring. Two experiments were performed using a transgenic (TF) and a non-transgenic (NTF) female. In experiment 1, three estrous cycles were monitored for the following parameters: estrus behavior, progesterone concentration and ovarian activity. In experiment 2, two superovulation/embryo recovery sessions were performed and the recovered embryos were transferred to previously prepared recipients. Data were compared by either t test or Fisher's exact test. The mean interval between natural estrus was 20.7 ± 0.6 and 19.7 ± 0.6 (P > 0.05) days for the TF and NTF, respectively. Progesterone concentrations and ovarian activity were normal and similar between goats. The ovulation rate was similar between TF and NTF (12.0 ± 1.4 vs. 18.0 ± 4.2 CL; P > 0.05). No significant differences in embryo recovery rate (P > 0.05) were observed between the surgical and transcervical methods for TF (69.2 vs. 72.7%) or NTF (100.0 vs. 86.7%). Sixteen embryos from the TF were transferred to recipients, and eight kids were born. Among these kids, the transgene was identified in three (two males and one female), resulting in a transgenesis rate of 37.5%. In summary, the TF is a true founder, since she proved fertility and capacity of transmitting the hG-CSF transgene to progeny, suggesting that the analyzed reproductive traits were not compromised by the presence of the transgene.

Reproductive parameters and the use of MOET in transgenic founder goat carrying the human granulocyte colony-stimulating factor (hG-CSF) gene

This study aimed to monitor estrous cycle parameters of a human granulocyte colony-stimulating factor (hG-CSF)-transgenic founder female goat and to perform superovulation and embryo recovery (surgical or transcervical method) for further transfer to recipients to quickly obtain offspring. Two experiments were performed using a transgenic (TF) and a non-transgenic (NTF) female. In experiment 1, three estrous cycles were monitored for the following parameters: estrus behavior, progesterone concentration and ovarian activity. In experiment 2, two superovulation/embryo recovery sessions were performed and the recovered embryos were transferred to previously prepared recipients. Data were compared by either t test or Fisher's exact test. The mean interval between natural estrus was 20.7 ± 0.6 and 19.7 ± 0.6 (P > 0.05) days for the TF and NTF, respectively. Progesterone concentrations and ovarian activity were normal and similar between goats. The ovulation rate was similar between TF and NTF (12.0 ± 1.4 vs. 18.0 ± 4.2 CL; P > 0.05). No significant differences in embryo recovery rate (P > 0.05) were observed between the surgical and transcervical methods for TF (69.2 vs. 72.7%) or NTF (100.0 vs. 86.7%). Sixteen embryos from the TF were transferred to recipients, and eight kids were born. Among these kids, the transgene was identified in three (two males and one female), resulting in a transgenesis rate of 37.5%. In summary, the TF is a true founder, since she proved fertility and capacity of transmitting the hG-CSF transgene to progeny, suggesting that the analyzed reproductive traits were not compromised by the presence of the transgene.(AU)

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil.

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.

Embryonic hybrid cells: a powerful tool for studying pluripotency and reprogramming of the differentiated cell chromosomes.

The properties of embryonic hybrid cells obtained by fusion of embryonic stem (ES) or teratocarcinoma (TC) cells with differentiated cells are reviewed. Usually, ES-somatic or TC-somatic hybrids retain pluripotent capacity at high levels quite comparable or nearly identical with those of the pluripotent partner. When cultured in vitro, ES-somatic- and TC-somatic hybrid cell clones, as a rule, lose the chromosomes derived from the somatic partner; however, in some clones the autosomes from the ES cell partner were also eliminated, i.e. the parental chromosomes segregated bilaterally in the ES-somatic cell hybrids. This opens up ways for searching correlation between the pluripotent status of the hybrid cells and chromosome segregation patterns and therefore for identifying the particular chromosomes involved in the maintenance of pluripotency. Use of selective medium allows to isolate in vitro the clones of ES-somatic hybrid cells in which "the pluripotent" chromosome can be replaced by "the somatic" counterpart carrying the selectable gene. Unlike the TC-somatic cell hybrids, the ES-somatic hybrids with a near-diploid complement of chromosomes are able to contribute to various tissues of chimeric animals after injection into the blastocoel cavity. Analysis of the chimeric animals showed that the "somatic" chromosome undergoes reprogramming during development. The prospects for the identification of the chromosomes that are involved in the maintenance of pluripotency and its cis- and trans-regulation in the hybrid cell genome are discussed.

Genetic modification of mammalian genome at chromosome level.

The review is concerned with a progress in genetic modification of a mammalian genome in vitro and in vivo at chromosomal level. Recently three new approaches for the chromosome biotechnology have been developed: Using Cre/loxP-system a researcher is able to produce targeted rearrangements of whole chromosomes or their segments or particular genes within the genome, and therefore to modify the set, position and copy number of the endogenous elements of the genome. Mammalian artificial chromosomes (MACs) provide a possibility to introduce into genome relatively large segments of alien chromosome material, either artificially constructed or derived from the genome of different species. Using ES-somatic cell hybrids allows to transfer whole chromosomes or their fragments between different genomes within and between species. Advantages and limitations of these approaches are discussed.