Dataset Information

Transcriptome Analysis of Pig In Vivo, In Vitro-Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway.

ABSTRACT: Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro-fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SAHA (10??M) had the highest level of blastocyst development at 43.9%, and all treatments except 10??M ISAHA had the same percentage of blastocyst development as Scriptaid (p<0.05). Two treatments, 1.0??M ISAHA and 1.0??M SAHA, had higher mean cell number than No HDACi treatment (p<0.021). Embryo transfers performed with 10??M SAHA- and 1??M ISAHA-treated embryos resulted in the birth of healthy piglets. GenBank accession numbers from up- and downregulated transcripts were loaded into the Database for Annotation, Visualization and Integrated Discovery to identify enriched biological themes. HDACi treatment yielded the highest enrichment for transcripts within the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway, lysosome. The mean intensity of LysoTracker was lower in IVV embryos compared to IVF and SCNT embryos (p<0.0001). SAHA and ISAHA can successfully be used to create healthy piglets from SCNT.

SUBMITTER: Whitworth KM

PROVIDER: S-EPMC4529055 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Publications

Transcriptome Analysis of Pig In Vivo, In Vitro-Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway.

Whitworth Kristin M KM Mao Jiude J Lee Kiho K Spollen William G WG Samuel Melissa S MS Walters Eric M EM Spate Lee D LD Prather Randall S RS

Cellular reprogramming 20150801 4

Genetically modified pigs are commonly created via somatic cell nuclear transfer (SCNT). Treatment of reconstructed embryos with histone deacetylase inhibitors (HDACi) immediately after activation improves cloning efficiency. The objective of this experiment was to evaluate the transcriptome of SCNT embryos treated with suberoylanilide hydroxamic acid (SAHA), 4-iodo-SAHA (ISAHA), or Scriptaid as compared to untreated SCNT, in vitro-fertilized (IVF), and in vivo (IVV) blastocyst-stage embryos. SA ...[more]

PMID: 26731590

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Project description:Somatic cell nuclear transfer and transcription factor overexpression can induce reprogramming of somatic cells, whereby one cell fate is changed into another cell fate of choice. Yet the efficiency of this process for generating functional cells is low, limiting their therapeutic applications. The persistence of transcriptional memory from the cell's prior identity is thought to be a major hindrance to effective reprogramming and differentiation to functional cell types. To explore the effects of transcriptional memory on cell fate specification of reprogrammed cells, we analyzed epidermal tissue development in nuclear transfer embryos derived from endoderm nuclei. Our findings reveal variations in the success rate of differentiation to functional cells across cell types in cloned embryos. While some cell types, such as goblet cells, differentiated normally, a specific subset of cells resisted cell fate reprogramming, adopting a new endoderm-like state and disrupting normal body patterning. Furthermore, inefficient transcriptional reprogramming correlated with reduced basal stem cell populations, faulty differentiation of basal stem cell-derived fates, and increased cell death in the epidermis of cloned embryos. Crucially, we identified that the memory of active transcriptional states linked to key endoderm transcription factors plays a significant role in these issues. Mimicking active state transcriptional memory of these genes through the forced expression of Sox17b and Foxa4 in the epidermis of fertilized embryos produced the same defects. On the contrary, reducing transcriptional memory by interfering with the expression of Sox17b led to the rescue of observed epidermal defects. In summary, our study suggests that transcriptional memory tends to persist predominantly in specific reprogrammed cell types, hindering their differentiation into functional cells and embryonic structures. This underscores the critical need to assess and reduce transcriptional memory from the previous somatic identity during reprogramming, to efficiently generate functional cell types for therapeutic applications.

Project description:Combining somatic cell nuclear transfer (SCNT) with genome editing technologies has emerged as a powerful platform for the creation of unique swine lineages for agricultural and biomedical applications. However, successful application of this research platform is still hampered by the low efficiency of these technologies, particularly in attaining complete cell reprogramming for the production of cloned pigs. Treating SCNT embryos with histone deacetylase inhibitors (HDACis), such as Scriptaid, has been routinely used to facilitate chromatin reprogramming after nuclear transfer. While increasing histone acetylation leads to a more relaxed chromatin configuration that facilitates the access of reprogramming factors and DNA repair machinery, it may also promote the expression of genes that are unnecessary or detrimental for normal embryo development. In this study, we evaluated the impact of inhibiting both histone deacetylases and RNA synthesis on pre- and post-implantation development of pig SCNT embryos. Our findings revealed that transcription can be inhibited for up to 40 h of development in porcine embryos, produced either by activation, fertilization or SCNT, without detrimentally affecting their capacity to form a blastocyst and their average number of cells at this developmental stage. Importantly, inhibiting RNA synthesis during HDACi treatment resulted in SCNT blastocysts with a greater number of cells and more abundant transcripts for genes related to embryo genome activation on days 2, 3 and 4 of development, compared to SCNT embryos that were treated with HDACi only. In addition, concomitant inhibition of histone deacetylases and RNA synthesis promoted the full reprograming of somatic cells, as evidenced by the normal fetal and full-term development of SCNT embryos. This combined treatment may improve the efficiency of the genome-editing + SCNT platform in swine, which should be further tested by transferring more SCNT embryos and evaluating the health and growth performance of the cloned pigs.

Dataset Information

Transcriptome Analysis of Pig In Vivo, In Vitro-Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway.

Publications

Transcriptome Analysis of Pig In Vivo, In Vitro-Fertilized, and Nuclear Transfer Blastocyst-Stage Embryos Treated with Histone Deacetylase Inhibitors Postfusion and Activation Reveals Changes in the Lysosomal Pathway.

Similar Datasets

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