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Made available in DSpace on 2021-06-25T11:55:29Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-03-30 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) This study aims to describe the early development of Piaractus mesopotamicus in detail at 22 degrees C, 26 degrees C, and 30 degrees C and describe primordial germ cell migration in vivo by using artificial GFP-nos1 3'UTR mRNA for future application in surrogate technology. The highest hatching rate was observed at 26 degrees C with 52.9 +/- 18.9%, which was considered the best incubation temperature. At 22 degrees C, the embryos survived only until the blastula stage. The first visualization of PGCs occurred in the segmentation period when the embryos presented between 6 and 16 somites. The number of GFP-positive cells varied from 7 to 18 per embryo (mean 11.2; n = 15). After hatching, the GFP-positive cells ranged from 7 to 22 per embryo (mean 14.6; n = 15). The PGCs were traced until the 9th day after hatching. The obtained data allow for the incubation temperature manipulation and early development and traceability of PGCs, which are important for reproductive biotechniques, establishment of cryobanks, and subsequent reconstitution through a germline chimera. Sao Paulo State Univ, Inst Biosci, Botucatu, SP, Brazil Chico Mendes Inst Biodivers Conservat, Natl Ctr Res & Conservat Continental Fish, Lab Fish Biotechnol, Pirassununga, SP, Brazil Univ Fed Rural Rio De Janeiro, Inst Zootecn, Rio De Janeiro, Brazil Univ Los Llanos, Inst Acuicultura Los Llanos, Villavicencio, Colombia, Brazil Hokkaido Univ, Fac Fisheries Sci, Hakodate, Hokkaido, Japan Sao Paulo State Univ, Inst Biosci, Botucatu, SP, Brazil FAPESP: 2010/17429-1 FAPESP: 2011/116641

An in vitro culture system of chicken primordial germ cells (PGCs) has been recently developed, but the growth factor involved in the proliferation of PGCs is largely unknown. In the present study, we investigated the growth effects of chicken stem cell factor (chSCF) on the in vitro proliferation of chicken PGCs. We established two feeder cell lines (buffalo rat liver cells; BRL cells) that stably express the putative secreted form of chSCF (chSCF1-BRL) and membrane bound form of chSCF (chSCF2-BRL). Cultured PGC lines were incubated on chSCF1 or chSCF2-BRL feeder cells with fibroblast growth factor 2 (FGF2), and growth effects of each chSCF isoform were investigated. The in vitro proliferation rate of the PGCs cultured on chSCF2-BRL at 20 days of culture was more than threefold higher than those cultured on chSCF1-BRL cells and more than fivefold higher than those cultured on normal BRL cells. Thus, use of chSCF2-BRL feeder layer was effective for in vitro proliferation of chicken PGCs. However, the acceleration of PGC proliferation on chSCF2-BRL was not observed without FGF2, suggesting that chSCF2 would act as a proliferation co-factor of FGF2. We transferred the PGCs cultured on chSCF2-BRL cells to recipient embryos, generated germline chimeric chickens and assessed the germline competency of cultured PGCs by progeny test. Donor-derived progenies were obtained, and the frequency of germline transmission was 3.39%. The results of this study demonstrate that chSCF2 induces hyperproliferation of chicken PGCs retaining germline competency in vitro in cooperation with FGF2., Article, Journal of Reproduction and Development.62(2):143-149(2016)

Formation of germline chimeras by transfer of chicken primordial germ cells (PGCs) is one of the effective techniques for preservation and regeneration of genetic resources in chickens. This study attempted to form germline chimeras of Gaok chicken buy purifying circulated PGCs of donor embryo before it is transferred to the recipient (White Leghorn chickens=WL) and studied the ability of recipient embryo on survival in incubators, and hatchability. This study used 200 fertile eggs of Gaok and 90 fertile WL breed all of the eggs was incubated at 38 0 C and 60% humidity in a portable incubator. PGCs-circulation of the blood collected Gaok embryos at stage 14-16 were taken from the dorsal aorta, and then purified by centrifugation method using nycodenz. PGCs-circulation results further purification frozen in liquid nitrogen before being transferred to the recipient embryo. The results showed that for the development of embryos transferred to the fresh circulation of PGCs-circulation as many as 25 cells can survive up to day 14, while one of the transferred of 50 and 100 cells into recipient embryos was hatched (10%). On the contrari recipient embryos that are transferred to the frozen PGCs-circulation the embryos development was shorter, and only survived until day 10th (treatment 25 cells), day 14th (treatment of 50 cells) and day 17th (treatment of 100 cells). It is concluded that the amount of PGCs-circulation embryos transferred to the recipient is one factor that influence the success of the development germline chimeras. Key Words: Gaok Chicken, White Leghorn Chicken, Circulated PGCs, Transfer, Germline Chimera

Transplantation of primordial germ cells (PGCs), which are the progenitor cells of gametes, is a powerful tool for generation of transgenic chickens. However, the frequencies of transgene integration into the genome of purified PGCs still remain low. An in vitro culture system enabling chicken PGCs to propagate efficiently would be useful for efficient transgenesis of PGCs. In the present study, we optimized the culture conditions for chicken PGCs to enhance the proliferation and evaluated the germline transmission of cultured PGCs that proliferated for long periods of time. PGC-like cells (PGC-LCs), that have remarkably similar morphological characteristics to intact PGCs, could be derived by cultivation of blood containing PGCs obtained from 2.5-day-old chicken embryos according to the protocol of van de Lavoir et al. (2006). We determined which feeder cells and which growth factors were required to improve proliferation of PGC-LCs. Male PGC-LCs survival and proliferation were enhanced during culture in the basic medium containing either basic fibroblast growth factor (bFGF) alone or both bFGF and stem cell factor (SCF) on a feeder of buffalo rat liver (BRL) cells. Male PGC-LCs could be propagated in defined culture condition for extended periods. These cells expressed the germline-specific protein Vasa and undifferentiated cell marker stage-specific embryonic antigen-1 (SSEA-1) and pluripotency genes Nanog and PouV. Furthermore, Male PGC-LCs cultured for 225 d could migrate toward and colonize within recipient gonads and transmit to the next generation following transplantation. We succeeded in produce 3 offspring originating from long-term cultured PGC-LCs from a germline chimeric rooster (6%). The present study represents valuable steps toward defining a culture condition enabling PGC-LCs to propagate efficiently for long periods in vitro with maintenance of their commitment to the germline., Article, JOURNAL OF POULTRY SCIENCE. 51(1):87-95 (2014)

The Japanese quail (Coturnix japonica) is a valuable bird as both an experimental animal, for a wide range of scientific disciplines, and an agricultural animal, for the production of eggs and meat. Cryopreservation of PGCs would be a feasible strategy for the conservation of both male and female fertility cells in Japanese quail. However, the effects of freeze-thaw treatment on viability, migration ability and germline transmission ability of quail PGCs still remain unclear. In the present study, male and female PGCs were isolated from the blood of 2-day-old embryos, which were cooled by slow freezing and then cryopreserved at –196 C for 77–185 days, respectively. The average recovery rate of PGCs after freeze-thawing was 47.0%. The viability of PGCs in the frozen group was significantly lower than that of the control group (P

Germ cell differentiation in reverse-sexed reproductive organs and interspecies germ line chimeras provides insight into the mechanism of germ cell development and represents a useful tool for conservation of endangered birds. We investigated the migration and survival capacity of male chicken primordial germ cells (PGCs) in female chicken embryos and in quail and Korean ring-necked pheasant embryos of both sexes. Interestingly, the PGCs were successfully reintroduced in all cases. Furthermore, the cells survived in the recipient gonads until hatching regardless of sex and species of the recipient. In the case of male recipient chickens, PGC-derived offspring were produced. However, the reverse-sexed female chickens, quails and pheasants of both sexes did not generate any male donor PGC-derived progeny. These results suggest that migration and survival circuits in chicken PGCs are conserved in both sexes and between avian species during embryonic development.

Over the past two decades numerous reports have demonstrated that the genetic modification of poultry genomes has great potential for improving poultry production; moreover, it may be used as a powerful tool for the production of industrial proteins. To date, transgenic techniques have been established for generating transgenic birds that express recombinant human proteins in hen eggs, as well as tissue-specific genes as an animal model. The production of transgenic birds is a promising approach that could have practical applications in agriculture and biopharmacology, in addition to advancing our understanding of avian biology. Finally, germ cell–mediated transgenesis could provide a more efficient strategy for creating gene-targeted insertions and deletions in avian species.

In previous studies, two types of offspring were generated from germline chimera between NH-413 strain (donor) and White Leghorn L-M strain (recipient). Phenotype and symptom of type-I offspring were quite similar to that of the NH-413 strain. In the other offspring of type-II, feather color showed mixture of white and brown and the symptom was not dominantly indicated. In the present studies, sexually matured males and females of the type-l were mated each other. Form these mating. chickens manifesting completely same phenotype to that of the donor NH-413 strain; brown feather color and symptoms of Muscular dystrophy. were regenerated. Therefore, complete regeneration of the muscular dystrophy chickens Could be achieved by mating males and female offspring derived from the germline chimeras. Fertility, hatchability and Survival rate of these regenerated offspring were significantly improved as compared to that of the original NH-413 strain. The established strategies should be one of the useful systems to regenerate chickens with muscular dystrophy., Article, JOURNAL OF POULTRY SCIENCE. 46(2): 123-126(2009)

A novel strategy has been developed to generate muscular dystrophy chickens by means of germline chimeras. Donor embryos were obtained from the New Hampshire chicken; NH-413 strain which have genes responsible for Fukuyama type muscular dystrophy (Saito et al., 2005). Donor cells were isolated from the center of area pellucida of the blastoderms. Recipient embryos were obtained from White Leghorn chicken; Line-M. The generated chimeric chickens had the donor derived brown plumage in the down in some extent, suggesting that the cells containing muscular dystrophy were introduced into the chimeras. These chimeric chickens have been raised until sexual maturity. The chimeric chickens were back-crossed to donor strain; the NH-413 strain. The phenotype of some of the offspring was very similar to that of the donor strain. The offspring showed some characters typical to the muscular dystrophy. It was suggested that the donor derived NH-413 strain offspring was generated. The established system should be one of the powerful strategies for breeding and regeneration of the muscular dystrophy chickens., Article, JOURNAL OF POULTRY SCIENCE. 46(1): 46-51(2009)

We report, for the first time, a series of baseline techniques comprising isolation and transplantation of female and male early-stage germ cells in sturgeon to generate a germline chimera as a potential tool for surrogate reproduction and gene banking. Cells were dissociated from testis, characterized by mostly spermatogonia, and from ovary, exclusively comprising oogonia and previtellogenic oocytes, of Acipenser baerii, using 0.3% trypsin (2 hours, 23 °C) dissolved in PBS, isotonic with blood plasma. The dissociated germ cells were sorted by Percoll gradient centrifugation followed by immunolabeling with germ cell–specific vasa antibody DDX4, while 10% to 30% Percoll solution contained 79.4% and 70.8% labeled testicular and ovarian cells. Sorted germ cells were transplanted into a cavity close to a presumptive genital ridge of newly hatched heterospecific Acipenser ruthenus larvae with fluorescein isothiocyanate–labeled endogenous primordial germ cells. The transplanted germ cells were randomly distributed in the body cavity through 30-day posttransplantation (dpt). Subsequently, the cells were organized into genital ridges 50 dpt and proliferated 90 dpt. The number of both transplanted and endogenous germ cells significantly increased from 18.1, 22.2, and 29.1 (30 dpt) to 108.5, 90.8, and 118.5 (90 dpt) in ovarian, testicular, and endogenous germ cells, respectively (P