Production Of Chimeras Research Articles

Transmission of mutant phenotypes from ES cells to adult mice.

Genetic manipulation of embryonic stem (ES) cells has been used to produce genetically engineered mice modeling human disorders. Here we describe a novel, additional application: selection for a phenotype of interest and subsequent transmission of that phenotype to a living mouse. We show, for the first time, that a cellular phenotype induced by ENU mutagenesis in ES cells can be transmitted and recapitulated in adult mice derived from these cells. We selected for paraquat-resistant (PQ(R)) ES clones. Subsequent injection of these cells into blastocysts resulted in the production of germline chimeras, from which tail skin fibroblasts exhibited enhanced PQ(R). This trait was also recovered in progeny of the chimera. We avoided PQ toxicity, which blocks the ability to involve the germline, by developing a sib-selection method, one that could be widely applied wherever the selection itself might diminish the pluripotency of the ES cells. Thus, phenotype-driven screens in ES cells are both feasible and efficient in producing intact mouse models for in vivo studies.

The reversible developmental unipotency of germ cells in chicken

We recently developed bimodal germline chimera production approaches by transfer of primordial germ cells (PGCs) or embryonic germ cells (EGCs) into embryos and by transplantation of spermatogonial stem cells (SSCs) or germline stem cells (GSCs) into adult testes. This study was undertaken to investigate the reversible developmental unipotency of chicken germ cells using our established germline chimera production systems. First, we transferred freshly isolated SSCs from adult testis or in vitro cultured GSCs into stage X and stage 14-16 embryos, and we found that these transferred SSCs/GSCs could migrate to the recipient embryonic gonads. Of the 527 embryos that received SSCs or GSCs, 135 yielded hatchlings. Of 17 sexually mature males (35.3%), six were confirmed as germline chimeras through testcross analysis resulting in an average germline transmission efficiency of 1.3%. Second, PGCs/EGCs, germ cells isolated from embryonic gonads were transplanted into adult testes. The EGC transplantation induced germline transmission, whereas the PGC transplantation did not. The germline transmission efficiency was 12.5 fold higher (16.3 vs 1.3%) in EGC transplantation into testis (EGCs to adult testis) than that in SSC/GSC transfer into embryos (testicular germ cells to embryo stage). In conclusion, chicken germ cells from different developmental stages can (de)differentiate into gametes even after the germ cell developmental clock is set back or ahead. Use of germ cell reversible unipotency might improve the efficiency of germ cell-mediated germline transmission.

Hematopoietic stem cell engraftment by early-stage in utero transplantation in a mouse model

A novel intrauterine transplantation (IUT) approach was developed to improve the efficiency of engraftment of hematopoietic stem cells (HSCs). HSCs with a green fluorescent protein (GFP) reporter gene were transplanted in utero on days 12.5, 13.5 and 14.5 post coitum (p.c.). The degree of chimerism of donor cells in recipient newborn mice was examined using fluorescent microscopy, polymerase chain reaction (PCR), fluorescence-activated cell sorting (FACS), and fluorescence in situ hybridization (FISH) analyses. Microscopic examination revealed the presence of green fluorescent signal in the peripheral blood of the chimeric mice. The highest survival rate (47%) as well as the highest chimerism rate (73%) were achieved by our new approach in the newborn mice that were subjected to in utero transplantation (IUT) on day 12.5 p.c. (E12.5) compared to the conventional IUT method. FACS analysis indicated that 1.55 ± 1.10% of peripheral blood cells from the newborn mice were GFP-positive donor cells. FISH showed that cells containing the donor-specific GFP sequence were present in the bone marrow (BM) of the chimeric mice. Thus, the efficiency of chimera production with this new method of IUT was significantly improved over the existing IUT techniques and instruments.

Counting Chromosomes in Embryonic Stem (ES) Cells

INTRODUCTIONAneuploidy in embryonic stem (ES) cells is the major cause of failure in obtaining contributions to all tissues of chimeras, including the germ line. This protocol describes a simple method for counting chromosomes from metaphase spreads in ES cells. ES cell clones showing euploidy on 70%-80% of spreads are used for chimera production.

Morphology and cytology of flower chimeras in hybrids of Brassica carinata ◊ Brassica rapa

Hybridization between white flowered Brassica carinata and yellow flowered B. rapa were made, and the flower chimeras were observed in a few hybrids. The simple single sequence molecular markers verified the hybridity of those hybrids. Chimeras were justified and totally classified based on the morphological characteristics of the flower petals that appeared in the hybrids of B. carinata and B. rapa. Two kinds of flower chimeras were observed: one type was different flower petals were with different colour in one branch; another type was that yellow petals were with white variegations, but the variegation size and shape were different in different petals. The meiosis and mitosis analysis showed that the partial or complete separation of parental genomes inferred to occur in pollen mother cells, shoot and early-developed petals in the flower chimeral hybrids, which hinted that the occurrence of complete or partial segregation of parental genomes in the somatic cells might be the reason for the production of flower chimera in the hybrids of B. carinata and B. rapa.

Depletion of Primordial Germ Cells (PGCs) by X-irradiation to Extraembryonic Region of Chicken Embryos and Expression of Xenotransplanted Quail PGCs

The generation of germline chimeras by the transfer of primordial germ cells (PGCs) requires incorporation of the PGCs of the donor into the gonadal tissue of the recipient embryo. We investigated the utility of soft x-irradiation with application of a lead (12×3×0.25mm, ∼0.1g) shield to the embryo proper for the production of chicken-quail germline chimeras. Chicken embryos shielded during irradiation for 120 s (∼7.2Gy) at stages 13 to 17 showed a hatchability of 35% (106/301), whereas the hatchability of unshielded embryos was 26% (27/105). The relative population of gonadal PGCs at stage 30 for embryos irradiated at stage 13 with or without shielding was 13 and 5%, respectively, of the value for nonirradiated controls. Chicken embryos irradiated at stages 13 or 14 with or without shielding and transfused with quail embryonic blood containing PGCs each exhibited ∼130 relative population of donor PGCs in the left gonad at stage 30. Xenotransplanted hatchlings exhibited donor-derived PGCs as detected by Southern hybridization and PCR. Exposure of chicken embryos to ∼7.2Gy of x-radiation at stage 13 with the application of a lead shield to the embryo proper is thus a feasible approach to depletion of endogenous germ cells and the production of chicken-quail germline chimeras.

Germline competence of mouse ES and iPS cell lines: Chimera technologies and genetic background

In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knock-out"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly strain-dependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.

The Bioactivity and Receptor Affinity of Recombinant Tagged EGF Designed for Tissue Engineering Applications Is Defined by the Nature and Position of the Tags

For tissue engineering applications, growth factor immobilization on cell culture scaffolds bears the potential to stimulate cell proliferation while minimizing costs associated to soluble growth factor supply. In order to evaluate the potential of a de novo-designed heterodimerization peptide pair, namely the E and K coils, for epidermal growth factor (EGF) grafting on various scaffolds, production of coil-tagged EGF chimeras using a mammalian cell expression system as well as their purification have been performed. The influence of the type of coil (E or K) upon EGF bioactivity, assessed in an in vitro cell assay, was compared to that of the fragment crystallizable (Fc) domain of immunoglobulin G by monitoring phosphorylation of EGF receptor (EGFR) upon chimeric EGF exposure. Our results demonstrate that the type and the location of the tag have a strong impact on growth factor bioactivity (EC50 ranging from 5.5 to 63 nM). Additional surface plasmon resonance-based biosensor experiments were conducted to test the ability of captured chimeric EGF to bind to their receptor ectodomain in vitro. These experiments indicated that the oriented coiled-coil-mediated immobilization of EGF was significantly more efficient than a random approach as coil-tagged EGF displayed enhanced affinities for artificially dimerized EGFR ectodomain when compared to Fc-tagged EGF (apparent KD of 5 pM vs. 16 nM). Altogether, our results highly suggest that coil-tagged chimeras represent an attractive avenue for the oriented immobilization of growth factors for tissue engineering applications and that HEK293 cells offer a robust platform for their expression in a bioactive form.

Cell Intrinsic TGF-β1 Regulation of B Cells

TGF-beta family cytokines play multiple roles in immune responses. TGF-beta1-null mice suffer from multi-organ infiltration that leads to their premature death. T cells play a central role in the TGF-beta1 phenotype, as deficiency of TGF-beta1 only in T cells reproduces the lethal phenotype. Although it is known that TGF-beta1 controls B cells isotype switch and homeostasis, the source responsible for this control has not been characterized. Because of the major role that T cells play in regulating B cell responses, we addressed the T cell dependency of the TGF-beta1 control of B cells. The analysis of T cell-deficient, TGF-beta1 knockout mice and the production of chimeras in which B but not T cells lacked TGF-beta1 allowed us to show that B cells are controlled in part by cell autonomous production of TGF-beta1.

Embryonic Stem Cells Contribute to Mouse Chimeras in the Absence of Detectable Cell Fusion

Embryonic stem (ES) cells are capable of differentiating into all embryonic and adult cell types following mouse chimera production. Although injection of diploid ES cells into tetraploid blastocysts suggests that tetraploid cells have a selective disadvantage in the developing embryo, tetraploid hybrid cells, formed by cell fusion between ES cells and somatic cells, have been reported to contribute to mouse chimeras. In addition, other examples of apparent stem cell plasticity have recently been shown to be the result of cell fusion. Here we investigate whether ES cells contribute to mouse chimeras through a cell fusion mechanism. Fluorescence in situ hybridization (FISH) analysis for X and Y chromosomes was performed on dissociated tissues from embryonic, neonatal, and adult wild-type, and chimeric mice to follow the ploidy distributions of cells from various tissues. FISH analysis showed that the ploidy distributions in dissociated tissues, notably the tetraploid cell number, did not differ between chimeric and wild-type tissues. To address the possibility that early cell fusion events are hidden by subsequent reductive divisions or other changes in cell ploidy, we injected Z/EG (lacZ/EGFP) ES cells into ACTB-cre blastocysts. Recombination can only occur as the result of cell fusion, and the recombined allele should persist through any subsequent changes in cell ploidy. We did not detect evidence of fusion in embryonic chimeras either by direct fluorescence microscopy for GFP or by PCR amplification of the recombined Z/EG locus on genomic DNA from ACTB-cre::Z/EG chimeric embryos. Our results argue strongly against cell fusion as a mechanism by which ES cells contribute to chimeras.

Restriction of Germline Proliferation by Soft X-ray Irradiation of Chicken Embryos and its Application to Chimera Production

Primordial germ cells (PGCs) are the progenitor cells of gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. PGCs enter the circulation soon after the formation of blood vessels in incubating eggs and eventually settle in the gonadal primordium. We have now examined exposure of chicken embryos to soft (low-energy) x-rays as a means of depleting endogenous PGCs and thereby improving the efficiency of chimera production. The blastoderm of White Leghorn eggs was exposed to soft x-rays for 0, 20, 40 or 60s before incubation. The irradiated embryos manifested delayed development at 60h of incubation. They also showed reduced numbers of circulating PGCs at stages 14 and 15 and of gonadal PGCs at stage 30. The hatchability of irradiated embryos was lower than that of nonirradiated controls. Irradiation for 20s was found to provide the best outcome taking into consideration both the restriction of PGC proliferation and hatchability. Dispersed blastoderm cells of quail (black plumage) embryos were introduced into the blastoderm of chicken embryos irradiated for 20 s or of nonirradiated embryos. The number of donor-derived PGCs was higher in the irradiated embryos than in the nonirradiated controls at stage 30. These results suggest that soft x-irradiation of chicken embryos is a feasible approach to depletion of endogenous germ cells and consequent improvement in the efficiency of incorporation of donor PGCs.

The Lipid Droplet Binding Domain of Hepatitis C Virus Core Protein Is a Major Determinant for Efficient Virus Assembly

Hepatitis C virus core protein forms the viral capsid and is targeted to lipid droplets (LDs) by its domain 2 (D2). By using a comparative analysis of two hepatitis C virus genomes (JFH1 and Jc1) differing in their level of virus production in cultured human hepatoma cells, we demonstrate that the core of the genotype 2a isolate J6 that is present in Jc1 mediates efficient assembly and release of infectious virions. Mapping studies identified a single amino acid residue in D2 as a major determinant for enhanced assembly and release of infectious Jc1 particles. Confocal microscopy analyses demonstrate that core protein in JFH1-replicating cells co-localizes perfectly with LDs and induces their accumulation in the perinuclear area, whereas no such accumulation of LDs and only a partial co-localization of core and LDs were found with the Jc1 genome. By using a fluorescence recovery after photobleaching assay, we found that green fluorescent protein-tagged D2 variants are mobile on LDs and that J6- and JFH1-D2 differ in their mobility. Taken together, our results demonstrate that the binding strength of the D2 domain of core for LDs is crucial for determining the efficiency of virus assembly.

Production of quail (Coturnix japonica) germline chimeras derived from in vitro‐cultured gonadal primordial germ cells

A previous report from our laboratory documented successful production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells (gPGCs). Subsequently, this study was designed to evaluate whether gPGCs can be maintained in vitro for extended period, and furthermore, these cultured PGCs can induce germline transmission after transfer into recipient embryos. In experiment 1, gonadal cells from the two strains (wild-type plumage (WP) and black (D) quail) were cultured in vitro for 10 days. Using antibody QCR1, we detected a continuous, significant (P = 0.0002) increase in the number of WP, but not D, PGCs. QCR1-positive WP colonies began to form after 7 days in culture. On Day 10 of culture, 803 WP PGCs were present as a result of a continuous increase, whereas no D PGC colonies could be detected and the D gonadal stroma cells were rolled up. Differences in the PGCs or the gonadal stroma cells of the two different strains might account for these differences. In experiment 2, WP PGC colonies were maintained in vitro up to Day 20 of culture, and 10- or 20-day-cultured PGCs were microinjected into dorsal aortas of 181 recipient D embryos. Thirty-five (19.3%) of the transplanted embryos hatched after incubation, and 25 (71.4%) of the hatchlings reached sexual maturity. Testcrossing of the sexually mature hatchlings resulted in three (10 days, 33.3%) and eight (20 days, 50.0%) germline chimeras respectively. This report is the first to describe successful production of germline chimera by transfer of in vitro-cultured gPGCs in quail.

Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background.

Na/K-ATPase β1 Subunit Expression Is Required for Blastocyst Formation and Normal Assembly of Trophectoderm Tight Junction-associated Proteins

Na/K-ATPase plays an important role in mediating blastocyst formation. Despite the expression of multiple Na/K-ATPase alpha and beta isoforms during mouse preimplantation development, only the alpha1 and beta1 isoforms have been localized to the basolateral membrane regions of the trophectoderm. The aim of the present study was to selectively down-regulate the Na/K-ATPase beta1 subunit employing microinjection of mouse 1 cell zygotes with small interfering RNA (siRNA) oligos. Experiments comprised of non-injected controls and two groups microinjected with either Stealthtrade mark Na/K-ATPase beta1 subunit oligos or nonspecific Stealthtrade mark siRNA as control. Development to the 2-, 4-, 8-, and 16-cell and morula stages did not vary between the three groups. However, only 2.3% of the embryos microinjected with Na/K-ATPase beta1 subunit siRNA oligos developed to the blastocyst stage as compared with 73% for control-injected and 91% for non-injected controls. Na/K-ATPase beta1 subunit down-regulation was validated by employing reverse transcription-PCR and whole-mount immunofluorescence methods to demonstrate that Na/K-ATPase beta1 subunit mRNAs and protein were not detectable in beta1 subunit siRNA-microinjected embryos. Aggregation chimera experiments between beta1 subunit siRNA-microinjected embryos and controls demonstrated that blockade of blastocyst formation was reversible. The distribution of Na/K-ATPase alpha1 and tight junction-associated proteins occludin and ZO-1 were compared among the three treatment groups. No differences in protein distribution were observed between control groups; however, all three polypeptides displayed an aberrant distribution in Na/K-ATPase beta1 subunit siRNA-microinjected embryos. Our results demonstrate that the beta1 subunit of the Na/K-ATPase is required for blastocyst formation and that this subunit is also required to maintain a normal Na/K-ATPase distribution and localization of tight junction-associated polypeptides during preimplantation development.

Pure line laying chickens at the Agassiz Research Centre

SummarySix lines of laying chickens representing high-producing non-industrial lines chosen or produced with consideration for characters of production are being kept at the Agassiz Research Centre. The collection includes one Barred Plymouth Rock (Line 60), one Columbian Plymouth Rock, one Rhode Island Red (Line 50), and three White Leghorn lines (Blue, Black, and Burgundy). Before coming to the Agassiz Research Centre these pure lines were subjected to mild selection for egg production and their egg production approaches that of commercial hybrids. The lines are currently maintained as a genetic resource as live populations without selection and with populations large enough to minimize inbreeding. In addition, samples of DNA from these lines have been conserved for genetic studies, and samples of embryonic cells are being kept cryogenically to allow the possibility of reconstitution of the lines through the production of germline chimeras. Techniques of ovarian transplantation are being developed which will allow more efficient cryogenic conservation and recuperation of the genetic material into live populations.

291 ENRICHMENT OF CARDIOMYOCYTES DERIVED FROM MURINE EMBRYONIC STEM CELLS TRANSFECTED OR NOT WITH BOVINE GROWTH HORMONE GENE (bGH)

Embryonic stem cells (ESC) are a pluripotent cell type that may be differentiated into cell lineages derived from the three embryonic layers. In animal reproduction biotechnologies, one of the main applications of ESC is their use as a vehicle for the transference of mutant alleles into animal genoma. Transformed ESC can be used for production of chimeras or transgenic clones, searching for economically favorable genetic conditions. The objective of the present study was to evaluate the biological effects of transfection, based on cardiomyocyte differentiation of the H106 mES cell line transfected or not with bGH (mESC and mESCbGH). Transfection was performed using the Ecdysone-Inducible Mammalian System (InVitrogen Brasil, Ltda., Sao Paulo, Brazil) (Buck 2003 Acta Sci. Vet. 31, 270–271 abst), by means of electroporation of pIND plasmids containing the geneticin resistance gene and bGH linked to ecdysone inducible promoter, and lipofection of pVgRXR plasmids containing the zeocin resistance gene. Cells were purified by zeocin and geneticin selection, and bGH cDNA was demonstrated by PCR. bGH expression was not induced in the present study. Cells were cultivated in suspension using the hanging drop technique (Hopfl 2004 Germ Cell Protocols 2, 79–98), in 20-µL drops containing mESC suspension (100 000 cells mL–1), divided into six groups: control mESC, dimethyl sulfoxide (DMSO) mESC, retinoic acid (RA) mESC, control mESCbGH, DMSO mESCbGH, and RA mESCbGH. Cells were resuspended in DMEM medium supplemented with 15% fetal calf serum, 0.1 mm 2-mercaptoethanol, 2 mm sodium pyruvate, 0.1 mm nonessential amino acids, 2 mm L-glutamine and 50 µg mL–1 streptomycin; DMSO groups were supplemented with 1% DMSO on Day 0, and RA groups were supplemented with 1 � 10–7 m all-trans-RA on Day 2, exchanging 10 µL medium containing 2 � 10–7 m all-trans-RA. After 5 days, formed embryoid bodies (EBs) were transfered to 96-well gelatin-coated adherent plates, in medium without DMSO or RA. Medium exchange was carried out each 48 h. Beating cells were evaluated on Day 14. Immunocytochemistry was performed on Day 17. Statistical analyses to measure differences between treatments were performed using the chi-square test. Approximately 50 structures were formed in each group. Both cell lines (mESC and mESCbGH) exibited beating EBs on Day 14 (control mESC: 22.9%; control mESCbGH: 50.9%), and in both lineages DMSO increased beating structures (mESC DMSO: 48.9% (P < 0.01); mESCbGH DMSO: 71.2% (P < 0.05)). RA was toxic to both lineages, and the EBs dissociated into small clumps of cells, which did not form any beating cells. Beating EBs expressed cardiac Troponin I marker, characteristic of cardiomyocytes. Both cell lines, before and after transfection, differentiated into cardiomyocytes, increased the beating cell rate when exposed to DMSO, but did not form beating cells when exposed to RA. This demonstrates that transfection did not cause any detrimental biological effects during cardiomyocyte differentiation.

Putative Embryonic Stem Cell Lines from Pig Embryos

Embryonic stem cells (ES cells) were first established in the mouse, and they represent a population of pluripotent, undifferentiated cells derived from early embryos that is capable of proliferating without any limitation in an undifferentiated state. These cells retain the ability to differentiate in vitro or in vivo into derivates of all three germ layers, and when injected into blastocysts, they can participate in the formation of all tissues, including gonads (germ-line chimeras). It is possible to transfect them with a gene of interest, and the resulting transgenic cell lines can also be used for production of chimeras. Unfortunately, mammalian germ-line chimeras that can carry an inserted gene into their progeny have only been produced in the mouse. Logically, before application of stem cell therapies into a human medicine, it is necessary to verify the efficiency and safety of these methods with an acceptable animal model. The pig is currently used as a very convenient animal for pre-clinical applications, and therefore establishment of porcine ES cell lines is highly needed; unfortunately, no convincing ES cell lines have been produced in this species (and other domestic animals) to date. In this article, we discuss the recent advances in this field, especially oriented on possible reasons and obstacles why derivation of porcine ES cell lines is still unsuccessful.

Inadvertent presence of pluripotent cells in monolayers derived from differentiated embryoid bodies

The therapeutic use of embryonic stem (ES)-derived cells is restricted by a risk of teratoma formation. To test the hypothesis that some cells with pluripotency characteristics remain following the differentiation of embryoid bodies (EB) into monolayer cells, we transformed mouse ES cells using constructs comprised of the mTert promoter coupled to green fluorescent protein. In this manner, EBs could be identified as showing gradually diminishing expression of the fluorescent marker as a consequence of differentiation. After 2 weeks of incubation, however, small groups of fluorescent cells remained in the differentiated monolayer. When these cells were isolated, cultured and expanded under ES cell culture conditions, they recovered their ES cell morphology (herein denoted ES-EB). We found by immunocytochemistry, reverse transcription-PCR and bisulphite analysis that despite the fact that some of these ES-EB lost their capacity to express some pluripotency markers characteristic of ES cells and undergo the epigenetic modification (hypo-methylation) of some retrotransposons (RT), after several passages in ES media, the cell colonies recovered their capacity for both pluripotency marker expression and RT methylation. Furthermore, when assessed for their ability to form chimeras, most ES-EB lines were unable to do so, although they recovered this potential for chimera production after some passages in ES cell media. Our results highlight the need for specific screening of differentiated cells before their therapeutic use and indicate that under adequate culture conditions, cells that loose their potential for expressing key markers of pluripotency can recover this fundamental embryonic stem cell property.

Production of Chick Germline Chimeras from Fluorescence-Activated Cell-Sorted Gonocytes

Modification of the chicken germline has been difficult, because it has been challenging to fractionate sufficient numbers of primordial germ cells for manipulation and implantation into developing embryos. A technique to enrich cell suspensions for primordial germ cells, using fluorescence-activated cell sorting (FACS), has recently been developed. The objective of the current study was to demonstrate that the FACS-enriched early embryonic gonocytes could fully participate in development of the germline. Therefore, cells were disassociated from stage 27 gonads, incubated with mouse anti-stage-specific embryonic antigen-1, which was detected with goat-antimouse IgM-fluorescein isothiocyanate, and the fluorescently labeled cells were sorted from the unlabeled cells using FACS. The isolated gonocyte population was injected into the blastoderm of unincubated stage X embryos, the germinal crescent of 3-d embryos, and into the circulation of stage 17 embryos that were pretreated with busulfan. Barred Plymouth Rock gonocytes were implanted exclusively into recipient White Leghorn embryos, and White Leghorn gonocytes were implanted exclusively into Barred Plymouth Rock recipient embryos. Embryos were cultured until hatch, and male putative chimeras were reared to sexual maturity. Germline chimerism was evaluated by observing feather color of the progeny. All injection methods resulted in germline chimeras demonstrating that FACS-sorted gonocytes can fully participate in development. Moreover, it was demonstrated that gonocytes isolated from stage 27 embryonic gonads can be introduced into embryos at an earlier stage of development, and the introduced gonocytes can fully participate in germline development.