Sex of donor cell and reprogramming conditions predict the extent and nature of imprinting defects in mouse iPSCs

ABSTRACTReprogramming of somatic cells into induced Pluripotent Stem Cells (iPSCs) is a major leap towards personalized approaches to disease modelling and cell-replacement therapies. However, we still lack the ability to fully control the epigenetic status of iPSCs, which is a major hurdle for their downstream applications. A sensible indicator for epigenetic fidelity is genomic imprinting, a phenomenon dependent on DNA methylation, which is frequently perturbed in iPSCs by yet unidentified reasons. By using a secondary reprogramming system with murine hybrid donor cells, we conducted a thorough imprinting analysis using IMPLICON in multiple female and male iPSCs generated under different culture conditions. Our results show that imprinting defects are remarkably common in mouse iPSCs causing dysregulation of the typical monoallelic expression of imprinted genes. Interestingly, the nature of imprinting defects depends on the sex of the donor cell and their respective response to culture conditions. Under serum-free conditions, male iPSCs show global hypomethylation at imprinted regions, whereas in serum conditions show focal hypermethylation at specific loci. In contrast, female iPSCs always exhibit hypomethylation defects regardless of culture conditions. These imprinting defects are more severe than the global changes in DNA methylation, highlighting the sensitivity of imprinting loci to current iPSC generation protocols. Our results reveal clear predictors underlying different types of imprinting defects in mouse iPSCs. This knowledge is essential to devise novel reprogramming strategies aiming at generating epigenetically faithful iPSCs.

Download Full-text

67 CELL TYPE AND CULTURE OVER TIME EFFECT DNA METHYLTRANSFERASE 1 EXPRESSION IN BOVINE DONOR FIBROBLAST CELLS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab67 ◽

2007 ◽

Vol 19 (1) ◽

pp. 151 ◽

Cited By ~ 2

Author(s):

K. Moore ◽

E. Wroclawska ◽

J. M. Kramer ◽

S. L. Goicoa

Keyword(s):

Dna Methylation ◽

Real Time ◽

Cell Lines ◽

Real Time Pcr ◽

Culture Conditions ◽

Fibroblast Cells ◽

Cell Type ◽

Donor Cells ◽

Dnmt1 Expression ◽

Over Time

Aberrant chromatin remodeling has been implicated in the low success rates achieved from cloned embryos. Following fertilization, DNA methylation within a normal embryo is rapidly reduced to a very low level and remains low until the 8–16 cell stage when DNA methylation once again increases. In contrast, the majority of cloned embryos fail to exhibit a similar methylation pattern. This may be due to somatic cell-associated DNMT1s keeping methylation high. However, attempts to chemically modify methylation patterns of donor cells prior to cloning have proven problematic. The objective of this study was to determine if a more natural approach, such as culture conditions, time in culture, and/or cell type, could alter DNMT1 expression in donor fibroblast cells. Two experiments were designed to meet these objectives. Donor fibroblast cell lines were produced from biopsies taken from male and female skin, ovaries, and testes, and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15&percnt; fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, and 0.1 mM β-mercaptoethanol, in a humidified environment of 5&percnt; CO2 in air, at 39°C. In Experiment 1, cell lines were maintained at 70&percnt; confluence to passage 4, 8, and 12, and analyzed by reverse transcription real-time PCR. In Experiment 2, cell lines were evaluated under 3 culture conditions: proliferating (70&percnt; confluence), serum-starved (0.5&percnt; FBS), and confluent (100&percnt;), and analyzed by reverse transcription real-time PCR. RNA was isolated from cell lines using Trizol reagent (Invitrogen, Carlsbad, CA, USA), reverse transcribed, and analyzed for DNMT1 expression using Taqman real-time PCR, with β-actin as the reference standard. All samples and no template controls were run in triplicate. Final quantitation was done using the comparative CT method, and relative DNMT1 expression was analyzed using one-way ANOVA followed by LS means multiple comparisons. Cell type and passage number had a significant effect on DNMT1 expression. Ovarian fibroblasts had an overall increase in expression in DNMT1 over time (P < 0.05), whereas male skin fibroblasts demonstrated an opposite trend (P = 0.05). Female skin fibroblasts and testes fibroblasts also had a decrease in DNMT1 expression over time, but only approached significance (P < 0.10). For Experiment 2, culture conditions tested did not affect DNMT1 expression for any except one skin cell line. In that case, proliferating cells had significantly higher DNMT1 than quiescent cells (P < 0.005). This research emphasizes the importance of donor cell type and culture effects over time on gene expression. These important aspects should be considered when selecting and growing donor cells to be utilized in somatic cell nuclear transfer. This project was supported by National Research Initiative Competitive Grant no. 2006-35203-16620 from the USDA Cooperative State Research, Education, and Extension Service and the Florida Agricultural Experiment Station.

Download Full-text

400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab400 ◽

2010 ◽

Vol 22 (1) ◽

pp. 356

Author(s):

K. J. Williams ◽

K. R. Bondioli ◽

R. A. Godke

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Stem Cells ◽

Chromatin Remodeling ◽

Primary Cultures ◽

Donor Cell ◽

Global Methylation ◽

Histone 3 ◽

Donor Cells

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

Download Full-text

36 RESTORATION OF TELOMERE LENGTH IN CLONED PIG EMBRYOS DURING EARLY EMBRYOGENESIS IS NOT DEPENDENT ON TELOMERE LENGTH AND TYPE OF DONOR CELLS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab36 ◽

2013 ◽

Vol 25 (1) ◽

pp. 166

Author(s):

T. Q. Dang-Nguyen ◽

S. Haraguchi ◽

S. Akagi ◽

T. Somfai ◽

M. Kaneda ◽

...

Keyword(s):

Telomere Length ◽

Embryonic Stem ◽

Donor Cell ◽

Cell Types ◽

Telomere Elongation ◽

Dependent Process ◽

Donor Cells ◽

Different Types ◽

Cloned Pig

Analyses on telomere length in cloned animals have revealed diverse results depending on the donor cell types. In mice and cattle, telomere length is reset during morula-blastocyst transition and the restoration is thought to be a telomerase-dependent process. However, it is still unknown whether the pattern of telomere elongation during this transition is dependent on donor cell types. In the present study, we examined the changes of telomere length during morula-blastocyst transition in cloned porcine embryos using different types of donor cell. Embryonic stem-like cells (ES), cumulus cells (C), fibroblasts at passages 7 and 10 (F7 and F10, respectively) were used as donor cells to produce NT embryos (ES, C, F7, and F10 groups, respectively). Telomere lengths of ES (35.8 ± 1.5 kb), C (24.4 ± 0.5 kb), P7 (18.7 ± 0.6 kb), and P10 (17.2 ± 0.1 kb) cells were significantly different. In contrast, cloned morulae in ES, C, F7, and F10 groups did not have any significant differences in telomere length (18.2 ± 0.3, 17.8 ± 0.7, 18.5 ± 0.3, and 18.4 ± 0.4 kb, respectively). Likewise, cloned blastocysts in ES, C, F7, and F10 groups had similar telomere length (22.3 ± 1.5, 23.5 ± 2.6, 20.2 ± 1.0, and 20.9 ± 1.0 kb, respectively). However, the telomere of the blastocyst was significantly longer (P < 0.05) compared with the morula in the respective group. Furthermore, relative telomerase activities of cloned morulae in ES, C, F7, and F10 groups (4.2 ± 0.4, 4.0 ± 0.5, 5.1 ± 0.4, and 4.9 ± 0.4, respectively) were significantly lower (P < 0.01) than those of cloned blastocysts in the same groups (8.2 ± 1.1, 8.6 ± 0.6, 12.5 ± 2.9, and 8.3 ± 1.1, respectively). The proportions of blastocysts in cloned embryos for ES, C, F7, and F10 groups (10.0 ± 5.2, 17.3 ± 2.9, 13.5 ± 2.9, and 13.1 ± 3.6%, respectively) did not significantly differ. Total cell numbers in blastocysts for ES, C, F7, and F10 groups (28.3 ± 2.9, 32.6 ± 3.6, 30.4 ± 3.1, and 27.4 ± 2.2, respectively) were not significantly different as well. In the present study, we found that the telomere elongation in cloned pig embryos occurs during morula-blastocyst transition. This is consistent with the previous findings in in vivo and in vitro fertilization and cloned embryos in cattle and mice. We also revealed that although different types of cells (ES, C, and F) or the same type of cells with different telomere length (F7 and F10) were used for NT, their resultant morulae and blastocysts had similar telomere length. This suggests that the telomere restoration during morula-blastocyst transition is independent of telomere length and type of donor cells. An increase in telomerase activity during morula-blastocyst transition indicates that the elongation of telomere length was likely a telomerase-dependent process. In conclusion, restoration of telomere length in cloned porcine embryos during morula-blastocyst transition was independent of telomere length and type of donor cells, and likely a telomerase-dependent process.

Download Full-text

60EPIGENETIC CHARACTERISTICS OF BOVINE AND CAPRINE EMBRYOS AND DONOR CELLS USED FOR NUCLEAR TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab60 ◽

2004 ◽

Vol 16 (2) ◽

pp. 152

Author(s):

E. Memili ◽

E. Behboodi ◽

H.M. Meade ◽

Y. Echelard

Keyword(s):

Dna Methylation ◽

Nuclear Transfer ◽

Great Part ◽

Donor Cell ◽

Fibroblast Cell ◽

Fluorescent Imaging ◽

Epifluorescence Microscopy ◽

Preimplantation Embryos ◽

Donor Cells ◽

Significant Difference

The molecular aspects of epigenetic events taking place in nuclear transfer (NT)-derived embryos are not well defined, but DNA methylation is known to be involved. One leading hypothesis is that the significant losses that occur during both pre- and post-implantation development are in great part due to improper epigenetic reprogramming. Aims of this study were to perform comparative quantitative analyses of the overall DNA methylation of bovine (both IVF- and NT-derived) and caprine IVF-derived preimplantation embryos as well as of donor cells used for NT. Caprine IVF was performed according to Blash S et al. (2001 Theriogenology 54, 899–905). Bovine and caprine 8- to 16-cell embryos were harvested on Day 4 post-insemination (dpi). Caprine donor cells (two adult fibroblast cell lines, T75-514 and F638) at the G1 phase of the cell cycle were prepared as previously described (Memili E et al. 2003 Theriogenology 59, 274 abst). Bovine cumulus cells were serum starved for four days prior to use and NT were performed as previously described (Echelard Y et al. 2002 Theriogenology 57, 779 abst). Embryos and the donor cells were vacuum-fixed onto 10-μm filters, and DNA methylation was determined by immunoassaying with a well-defined anti-5-methyl-cytosine antibody (Dean W et al. 2001 Prod Natl Acad Sci 98(24), 13734–13738) and fluorescent labeled anti-mouse IgG as secondary antibody following the protocol of Shi and Haaf (2002Mol Reprod Dev 63, 329–334). Fluorescent imaging was performed by epifluorescence microscopy. The methylation-specific signal was recorded digitally with a high-resolution charge-coupled devise camera, followed by analysis with the MetaMorph™ imaging software (Universal Imaging Corporation, Downingtown, PA, USA). These experiments showed a high level of heterogeneity in the methylation levels of bovine and caprine donor cells. However, caprine 8- to 16-cell IVF embryos exhibited similar levels of DNA methylation to their bovine IVF counterparts. Conversely, when the DNA methylation level of 97 bovine IVF nuclei was compared to that of 55 bovine NT embryonic nuclei, significant differences were found. Levels of signal intensities per nucleus were almost 9-fold greater for NT embryos (133 v. 15), a significant difference (P<0.01). In conclusion, donor cell populations with heterogeneous DNA methylation and incomplete reprogramming of DNA methylation in NT embryos are likely to be the underlying reasons for low level of successful NT embryonic and fetal development. Thus, designing NT protocols supporting better reprogramming may result in improvement since, in order for a successful embryonic development after NT, DNA methylation needs to be reprogrammed from a somatic cell pattern to an early embryonic pattern. Furthermore, similar levels of DNA methylation between caprine and bovine IVF embryos may be an indication of a similar pattern of embryonic gene expression between these species.

Download Full-text

Dynamic integration of enteric neural stem cells in ex vivo organotypic colon cultures

Scientific Reports ◽

10.1038/s41598-021-95434-4 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Georgina Navoly ◽

Conor J. McCann

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

Organotypic Culture ◽

Ex Vivo ◽

Donor Cell ◽

Colonic Tissue ◽

Cell Therapies ◽

Enteric Ganglia ◽

Donor Cells

AbstractEnteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1cre/+;R26RYFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP+ neurospheres were transplanted to C57BL6/J (6–8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease.

Download Full-text

Strategy to Establish Embryo-Derived Pluripotent Stem Cells in Cattle

International Journal of Molecular Sciences ◽

10.3390/ijms22095011 ◽

2021 ◽

Vol 22 (9) ◽

pp. 5011

Author(s):

Daehwan Kim ◽

Sangho Roh

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Stem Cell Research ◽

Pluripotent Stem Cells ◽

Embryonic Stem ◽

Culture Conditions ◽

Human Diseases ◽

Induced Pluripotent

Stem cell research is essential not only for the research and treatment of human diseases, but also for the genetic preservation and improvement of animals. Since embryonic stem cells (ESCs) were established in mice, substantial efforts have been made to establish true ESCs in many species. Although various culture conditions were used to establish ESCs in cattle, the capturing of true bovine ESCs (bESCs) has not been achieved. In this review, the difficulty of establishing bESCs with various culture conditions is described, and the characteristics of proprietary induced pluripotent stem cells and extended pluripotent stem cells are introduced. We conclude with a suggestion of a strategy for establishing true bESCs.

Download Full-text

Biological importance of OCT transcription factors in reprogramming and development

Experimental & Molecular Medicine ◽

10.1038/s12276-021-00637-4 ◽

2021 ◽

Author(s):

Kee-Pyo Kim ◽

Dong Wook Han ◽

Johnny Kim ◽

Hans R. Schöler

Keyword(s):

Stem Cells ◽

Transcription Factors ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Ectopic Expression ◽

Donor Cell ◽

Structural Components ◽

Oct4 Expression ◽

Reprogramming Factors ◽

Induced Pluripotent

AbstractEctopic expression of Oct4, Sox2, Klf4 and c-Myc can reprogram somatic cells into induced pluripotent stem cells (iPSCs). Attempts to identify genes or chemicals that can functionally replace each of these four reprogramming factors have revealed that exogenous Oct4 is not necessary for reprogramming under certain conditions or in the presence of alternative factors that can regulate endogenous Oct4 expression. For example, polycistronic expression of Sox2, Klf4 and c-Myc can elicit reprogramming by activating endogenous Oct4 expression indirectly. Experiments in which the reprogramming competence of all other Oct family members tested and also in different species have led to the decisive conclusion that Oct proteins display different reprogramming competences and species-dependent reprogramming activity despite their profound sequence conservation. We discuss the roles of the structural components of Oct proteins in reprogramming and how donor cell epigenomes endow Oct proteins with different reprogramming competences.

Download Full-text

Rabbit somatic cell cloning: effects of donor cell type, histone acetylation status and chimeric embryo complementation

Reproduction ◽

10.1530/rep.1.01206 ◽

2007 ◽

Vol 133 (1) ◽

pp. 219-230 ◽

Cited By ~ 73

Author(s):

Feikun Yang ◽

Ru Hao ◽

Barbara Kessler ◽

Gottfried Brem ◽

Eckhard Wolf ◽

...

Keyword(s):

Histone Acetylation ◽

Nuclear Transfer ◽

Donor Cell ◽

Epigenetic Mark ◽

Cell Type ◽

Developmental Potential ◽

Acetylation Status ◽

Donor Cells ◽

Donor Cell Type

The epigenetic status of a donor nucleus has an important effect on the developmental potential of embryos produced by somatic cell nuclear transfer (SCNT). In this study, we transferred cultured rabbit cumulus cells (RCC) and fetal fibroblasts (RFF) from genetically marked rabbits (Alicia/Basilea) into metaphase II oocytes and analyzed the levels of histone H3-lysine 9-lysine 14 acetylation (acH3K9/14) in donor cells and cloned embryos. We also assessed the correlation between the histone acetylation status of donor cells and cloned embryos and their developmental potential. To test whether alteration of the histone acetylation status affects development of cloned embryos, we treated donor cells with sodium butyrate (NaBu), a histone deacetylase inhibitor. Further, we tried to improve cloning efficiency by chimeric complementation of cloned embryos with blastomeres fromin vivofertilized or parthenogenetic embryos. The levels of acH3K9/14 were higher in RCCs than in RFFs (P<0.05). Although the type of donor cells did not affect development to blastocyst, after transfer into recipients, RCC cloned embryos induced a higher initial pregnancy rate as compared to RFF cloned embryos (40 vs 20%). However, almost all pregnancies with either type of cloned embryos were lost by the middle of gestation and only one fully developed, live RCC-derived rabbit was obtained. Treatment of RFFs with NaBu significantly increased the level of acH3K9/14 and the proportion of nuclear transfer embryos developing to blastocyst (49 vs 33% with non-treated RFF,P<0.05). The distribution of acH3K9/14 in either group of cloned embryos did not resemble that inin vivofertilized embryos suggesting that reprogramming of this epigenetic mark is aberrant in cloned rabbit embryos and cannot be corrected by treatment of donor cells with NaBu. Aggregation of embryos cloned from NaBu-treated RFFs with blastomeres fromin vivoderived embryos improved development to blastocyst, but no cloned offspring were obtained. Two live cloned rabbits were produced from this donor cell type only after aggregation of cloned embryos with a parthenogenetic blastomere. Our study demonstrates that the levels of histone acetylation in donor cells and cloned embryos correlate with their developmental potential and may be a useful epigenetic mark to predict efficiency of SCNT in rabbits.

Download Full-text

Umbilical Cord Tissue as a Source of Young Cells for the Derivation of Induced Pluripotent Stem Cells Using Non-Integrating Episomal Vectors and Feeder-Free Conditions

Cells ◽

10.3390/cells10010049 ◽

2020 ◽

Vol 10 (1) ◽

pp. 49

Author(s):

Aisha Mohamed ◽

Theresa Chow ◽

Jennifer Whiteley ◽

Amanda Fantin ◽

Kersti Sorra ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Umbilical Cord ◽

Pluripotent Stem Cells ◽

Blood Cells ◽

Growth Media ◽

Donor Cells ◽

Episomal Vectors ◽

Umbilical Cord Tissue ◽

Induced Pluripotent

The clinical application of induced pluripotent stem cells (iPSC) needs to balance the use of an autologous source that would be a perfect match for the patient against any safety or efficacy issues that might arise with using cells from an older patient or donor. Drs. Takahashi and Yamanaka and the Office of Cellular and Tissue-based Products (PMDA), Japan, have had concerns over the existence of accumulated DNA mutations in the cells of older donors and the possibility of long-term negative effects. To mitigate the risk, they have chosen to partner with the Umbilical Cord (UC) banks in Japan to source allogeneic-matched donor cells. Production of iPSCs from UC blood cells (UCB) has been successful; however, reprogramming blood cells requires cell enrichment with columns or flow cytometry and specialized growth media. These requirements add to the cost of production and increase the manipulation of the cells, which complicates the regulatory approval process. Alternatively, umbilical cord tissue mesenchymal stromal cells (CT-MSCs) have the same advantage as UCB cells of being a source of young donor cells. Crucially, CT-MSCs are easier and less expensive to harvest and grow compared to UCB cells. Here, we demonstrate that CT-MSCs can be easily isolated without expensive enzymatic treatment or columns and reprogramed well using episomal vectors, which allow for the removal of the reprogramming factors after a few passages. Together the data indicates that CT-MSCs are a viable source of donor cells for the production of clinical-grade, patient matched iPSCs.

Download Full-text

Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells

Development ◽

10.1242/dev.108.1.185 ◽

1990 ◽

Vol 108 (1) ◽

pp. 185-189 ◽

Cited By ~ 4

Author(s):

J.N. Petitte ◽

M.E. Clark ◽

G. Liu ◽

A.M. Verrinder Gibbins ◽

R.J. Etches

Keyword(s):

Inbred Line ◽

Cell Lineage ◽

Donor Cell ◽

Black Pigment ◽

Dominant Allele ◽

White Leghorn ◽

Sperm Dna ◽

Donor Cells ◽

White Leghorns ◽

Blastodermal Cells

Cells were isolated from stage X embryos of a line of Barred Plymouth Rock chickens (that have black pigment in their feathers due to the recessive allele at the I locus) and injected into the subgerminal cavity of embryos from an inbred line of Dwarf White Leghorns (that have white feathers due to the dominant allele at the I locus). Of 53 Dwarf White Leghorn embryos that were injected with Barred Plymouth Rock blastodermal cells, 6 (11.3%) were phenotypically chimeric with respect to feather colour and one (a male) survived to hatching. The distribution of black feathers in the recipients was variable and not limited to a particular region although, in all but one case, the donor cell lineage was evident in the head. The male somatic chimera was mated to several Barred Plymouth Rock hens to determine the extent to which donor cells had been incorporated into his testes. Of 719 chicks hatched from these matings, 2 were phenotypically Barred Plymouth Rocks demonstrating that cells capable of incorporation into the germline had been transferred. Fingerprints of the blood and sperm DNA from the germline chimera indicated that both of these tissues were different from those of the inbred line of Dwarf White Leghorns. Bands that were present in fingerprints of blood DNA from the chimera and not present in those of the Dwarf White Leghorns were observed in those of the Barred Plymouth Rocks.(ABSTRACT TRUNCATED AT 250 WORDS)

Download Full-text