Gene expression and development of early pig embryos produced by serial nuclear transfer

2009 ◽  
Vol 76 (6) ◽  
pp. 555-563 ◽  
Author(s):  
Xiaojun Xing ◽  
Luca Magnani ◽  
Kiho Lee ◽  
Chunmin Wang ◽  
Ryan A. Cabot ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Serial Nuclear Transfer

scholarly journals Preimplantation embryo programming: transcription, epigenetics, and culture environment

Reproduction ◽  
2008 ◽  
Vol 135 (2) ◽  
pp. 141-150 ◽  
Author(s):  
Veronique Duranthon ◽  
Andrew J Watson ◽  
Patrick Lonergan
Keyword(s):  
Gene Expression ◽  
Embryo Culture ◽  
Nuclear Transfer ◽  
Preimplantation Embryo ◽  
Later Life ◽  
Short Review ◽  
Basic Program ◽  
Preimplantation Development ◽  
Preimplantation Embryos ◽  
Negative Consequences

Preimplantation development directs the formation of an implantation- or attachment-competent embryo so that metabolic interactions with the uterus can occur, pregnancy can be initiated, and fetal development can be sustained. The preimplantation embryo exhibits a form of autonomous development fueled by products provided by the oocyte and also from activation of the embryo's genome. Despite this autonomy, the preimplantation embryo is highly influenced by factors in the external environment and in extreme situations, such as those presented by embryo culture or nuclear transfer, the ability of the embryo to adapt to the changing environmental conditions or chromatin to become reprogrammed can exceed its own adaptive capacity, resulting in aberrant embryonic development. Nuclear transfer or embryo culture-induced influences not only affect implantation and establishment of pregnancy but also can extend to fetal and postnatal development and affect susceptibility to disease in later life. It is therefore critical to define the basic program controlling preimplantation development, and also to utilize nuclear transfer and embryo culture models so that we may design healthier environments for preimplantation embryos to thrive in and also minimize the potential for negative consequences during pregnancy and post-gestational life. In addition, it is necessary to couple gene expression analysis with the investigation of gene function so that effects on gene expression can be fully understood. The purpose of this short review is to highlight our knowledge of the mechanisms controlling preimplantation development and report how those mechanisms may be influenced by nuclear transfer and embryo culture.

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

68 GENE EXPRESSION COMPARISONS BETWEEN BOVINE IN VIVO AND CLONED EMBRYOS PRODUCED BY THREE DIFFERENT METHODS

2006 ◽  
Vol 18 (2) ◽  
pp. 142
Author(s):  
N. Ruddock ◽  
K. Wilson ◽  
M. Cooney ◽  
R. Tecirlioglu ◽  
V. Hall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chromatin Remodeling ◽  
Nuclear Transfer ◽  
Expression Patterns ◽  
Experimental Models ◽  
Gene Expression Patterns ◽  
Imprinted Genes ◽  
Mammalian Embryo

Developmental pathways in the mammalian embryo are profoundly influenced by the epigenetic interaction of the environment and the genome. Loss of epigenetic control has been implicated in aberrant gene expression and altered imprinting patterns with consequence to the physiology and viability of the conceptus. Bovine somatic cell nuclear transfer (SCNT) is contingent on in vitro culture, and both SCNT and culture conditions are known to induce changes in embryonic gene expression patterns. Using these experimental models, this study compared gene expression of Day 7 cloned blastocysts created from three different SCNT protocols using the same cell line, with Day 7 in vivo blastocysts to elucidate mechanisms responsible for variations in phenotypic outcomes. SCNT methods included: (1) traditional SCNT by subzonal injection (SI); (2) handmade cloning (HMC); and (3) modified serial nuclear transfer (SNT), developed within the group. Four imprinted genes (Grb10, Ndn, Nnat, and Ube3a), four chromatin remodeling genes (Cbx1, Cbx3, Smarca4, and Smarcb1) and two genes implicated in polycystic liver disease (Prkcsh and Sec63) were analyzed in single blastocysts from each treatment (n = 5). All blastocysts expressed Actin, Oct-4 and Ifn-tau. All genes were sequence verified. Several genes were expressed ubiquitously across all groups, including Ndn, Ube3a, Cbx1, Cbx3, and Smarcb1. Interestingly, Grb10 was not expressed in two HMCs and one SNT blastocyst. Nnat was weakly expressed in one in vivo blastocyst and in the majority of cloned blastocysts in all groups. Prkcsh and Sec63 were expressed in all but one HMC blastocyst. While gene expression patterns were mostly maintained following SCNT, the imprinted genes Nnat and Grb10 showed instances of differential or abnormal expression in SCNT embryos. The chromatin remodeling genes were maintained in all SCNT treatments. Prkcsh and Sec63 were both absent in one HMC blastocyst, with implications for liver dysfunction, a condition previously reported in abnormal cloned offspring. The variable mRNA expression following SCNT provides an insight into genetic and environmental factors controlling implantation, placentation, organ formation, and fetal growth.

64 X-LINKED GENE EXPRESSION IN BOVINE (BOS TAURUS) MALE AND FEMALE IN VITRO- FERTILIZED AND SOMATIC CELL NUCLEAR TRANSFER-DERIVED BLASTOCYSTS

2006 ◽  
Vol 18 (2) ◽  
pp. 140
Author(s):  
M. Nino-Soto ◽  
G. Mastromonaco ◽  
P. Blondin ◽  
W. A. King
Keyword(s):  
Gene Expression ◽  
Developmental Stage ◽  
Somatic Cell ◽  
Dosage Compensation ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Regulatory Mechanisms ◽  
Linked Gene ◽  
Male And Female

Expression of some X-chromosome linked genes has recently been shown to be altered in bovine somatic cell nuclear transfer (SCNT) derived embryos (Wrenzycki et al. 2002 Biol. Reprod. 66, 127), implying that the regulatory mechanisms of X-linked transcription are affected by embryo in vitro production (IVP) methods. We analyzed the transcriptional pattern of X-linked genes (BIRC4, GAB3, HPRT1, MECP2, RPS4X, SLC25A6, and XIST) in bovine in vitro fertilized (IVF) and SCNT male and female blastocysts to determine X-inactivation status and changes resulting from IVP. We collected pools of male (n = 5 pools) and female (n = 3 pools) IVF-derived blastocysts (Bousquet et al. 1999 Theriogenology 51, 59) and male (n = 5 pools) and female (n = 3 pools) SCNT-derived blastocysts (Mastromonaco et al. 2004 Reprod. Domest. Anim. 39, 462). Each pool consisted of five blastocysts. Embryos were washed in phosphate buffered saline (PBS) + 0.1% polyvinyl alcohol (PVA), collected, and stored at -80�C. Total RNA was extracted with an Absolutely RNA Microprep kit (Stratagene, La Jolla, CA, USA), DNase I treated, and precipitated with isopropanol and linear acrylamide (Ambion, Inc., Austin, TX, USA) as a carrier. Reverse transcription was performed with Oligo-dT (Invitrogen, Burlington, Ontario, Canada) and Superscript II RT (Invitrogen). Transcript quantification was performed by quantitative real-time PCR using SYBR Green I (LightCycler system, Roche, Diagnostics, Laval, Quebec, Canada). Data analysis was performed with SAS (SAS Institute, Inc., Cary, SC, USA) using a mixed-model factorial ANOVA and with results presented as estimates of the median, ratios of estimates, and 95% confidence intervals with � = 0.05. IVF-derived male and female blastocysts possessed similar levels of the transcripts analyzed, suggesting successful dosage compensation at this developmental stage for embryos fertilized in vitro. XIST was not detected in male IVF embryos. GAB3 was not detected in any of the female groups and, in addition, HPRT1 transcripts were not detected in SCNT derived female embryos. Male and female SCNT-derived blastocysts possessed marked differences in their transcript levels, with males showing statistically significantly higher levels of BIRC4 and RPS4X and females possessing higher levels of MECP2 and SLC25A6 transcripts although differences between the latter two were not statistically significant. XIST was detected in both male and female SCNT blastocysts. We conclude that dosage compensation between male and female IVF blastocysts is achieved at this developmental stage for the transcripts examined. However, this pattern was markedly changed in the SCNT group, affecting especially female SCNT blastocysts, suggesting that the regulatory mechanisms of X-inactivation and X-linked gene expression are substantially altered in SCNT embryos probably due to aberrant epigenetic patterns and faulty genome reprogramming. We are currently analyzing X-linked transcription in male and female in vivo-derived blastocysts in order to compare this group with IVP-derived embryos. This work was funded by NSERC, CIHR, and CRC.

257 EXPRESSION OF DEVELOPMENTAL GENES IN PORCINE NUCLEAR TRANSFER EMBRYOS RECONSTRUCTED WITH BONE MARROW MESENCHYMAL STEM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 236
Author(s):  
B. Mohana Kumar ◽  
H.-F. Jin ◽  
J.-G. Kim ◽  
S. Balasubramanian ◽  
S.-Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Donor Cells ◽  
Fetal Fibroblasts ◽  
Marrow Mesenchymal Stem Cells

Abnormal gene expression is frequently observed in nuclear transfer (NT) embryos and is one of the suggested causes of the low success rates of this approach. Recent study has suggested that adult stem cells may be better donor cells for NT, as their less differentiated state may ease epigenetic reprogramming by the oocyte (Kato et al. 2004 Biol. Reprod. 70, 415-418). In the present study, we investigated the expression profile of some selected genes involved in the development of the pre-implantation embryos of in vivo- and NT-derived origin using bone marrow mesenchymal stem cells (MSCs) and porcine fetal fibroblasts (pFF) as donors. Isolated population of MSCs from porcine bone marrow were characterized by cell-surface antigen profile (CD13pos, CD105pos, CD45neg, and CD133neg) and by their extensive consistent differentiation to multiple mesenchymal lineages (adipocytic, osteocytic and chondrocytic) under controlled in vitro conditions (Pittenger et al. 1999 Science 284, 143-147). Primary cultures of pFF from a female fetus at <30 days of gestation were established. for NT, donor cells at 3-4 passages were employed. Embryos cloned from MSCs showed enhanced developmental potential compared to pFF cloned embryos, indicated by higher rates of blastocyst formation (15.3% � 4.8 and 9.0% � 3.9, respectively) and total cell number (31.5 � 7.2 and 20.5 � 5.4, respectively) in Day 7 blastocysts. Total RNA was extracted from pools (triplicates) of 10 embryos each of 8-cell, morula, and blastocyst stages of in vivo and NT origin using Dynabeads� mRNA DIRECT" kit (Dynal, Oslo, Norway). Reverse transcription was performed with a Superscript" III cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed on a Light cycler� using FastStart DNA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany). The expression profiles of genes involved in transcription (Oct-4, Stat3), DNA methylation (Dnmt1), de novo methylation (Dnmt3a), histone deacetylation (Hdac2), anti-apoptosis (Bcl-xL), and embryonic growth (Igf2r) were determined. The mRNA of H2a was employed to normalize the levels. Significant differences (P < 0.05) in the relative abundance of Stat3, Dnmt1, Dnmt3a, Bcl2, and Igf2r were observed in pFF NT embryos compared with in vivo-produced embryos, whereas embryos derived from MSCs showed expression patterns similar to those of in vivo-produced embryos. However, Oct-4 and Hdac2 revealed similar expression profiles in NT- and in vivo-produced embryos. These results indicate that MSC-derived NT embryos had enhanced embryonic development and their gene expression pattern more closely resembled that of in vivo-produced embryos. Hence, less differentiated MSCs may have a more flexible potential in improving the efficiency of the porcine NT technique. This work was supported by Grant No. R05-2004-000-10702-0 from KOSEF, Republic of Korea.

500. THE MANIPULATION OF THE EPIGENETIC MARK HISTONE 3 LYSINE 9 TRIMETHYLATION IN DONOR CELLS AND ITS EFFECTS ON THE DEVELOPMENT OF CLONED MOUSE EMBRYOS

2009 ◽  
Vol 21 (9) ◽  
pp. 101
Author(s):  
J. Antony ◽  
F. Oback ◽  
R. Broadhurst ◽  
S. Cole ◽  
C. Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Expression Profiles ◽  
Es Cells ◽  
Embryonic Stem ◽  
Histone Demethylase ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Epigenetic Marks ◽  
Donor Cells

To produce live cloned mammals from adult somatic cells the nuclei of these cells must be first reprogrammed from a very restricted, cell lineage-specific gene expression profile to an embryo-like expression pattern, compatible with embryonic development. Although this has been achieved in a number of species the efficiency of cloning remains very low. Inadequate reprogramming of epigenetic marks in the donor cells correlated with aberrant embryonic gene expression profiles has been identified as a key cause of this inefficiency. Some of the most common epigenetic marks are chemical modifications of histones, the main structural proteins of chromatin. A range of different histone modifications, including acetylation and methylation, exists and can be attributed to either repression or activation of genes. One epigenetic mark which is known to be very stable and difficult to remove during reprogramming is the trimethylation of lysine 9 in histone H3 (H3K9Me3). To test the hypothesis that H3K9Me3 marks are a major stumbling block for successful cloning we are attempting to remove these marks by overexpression of the H3K9Me3 specific histone demethylase, jmjd2b, in donor cells, prior to their use for nuclear transfer. We have engineered mouse embryonic stem (ES) cells for the tet inducible expression of a fusion protein with a functional jmjd2b or non-functional mutant jmjd2b histone demethylase. Approximately 94% and 88% of the cells can be induced for the expression of functional and mutant jmjd2b-EGFP in the respective ES cell lines. Immunofluorescence analyses have shown that induction of functional jmjd2b-EGFP results in an approximately 50% reduction of H3K9Me3 levels compared to non-induced cells and induced mutant jmjd2b-EGFP cells. The comparison of the in-vitro embryo development following nuclear transfer with induced and non-induced donor cells show significantly better overall development to blastocysts and morulae from induced donor cells with reduced H3K9Me3 levels.

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