Jie Lan

Methylation patterns in 5＇ terminal regions of pluripotency-related genes in bovine in vitro fertilized and cloned embryos

In order to investigate DNA methylation profiles of five pluripotency-related genes (Oct4, Sox2, Nanog, Rex1 and Fgf4) during bovine maternal to zygotic transition (MZT) in both in vitro fertilized (IVF) and nuclear transfer (NT) embryos, sodium bisulfite sequencing method was used to detect DNA methylation levels, accompanied by the statistical analysis of embryo developmental rates. The results showed that Oct4, Nanog, Rex1 and Fgf4 were respectively demethylated by 25.22% (P < 0.01), 3.84% (P > 0.05), 31.82% (P < 0.01) and 10% (P > 0.05) while Sox2 retained unmethylation during MZT in IVF embryos. By contrast, Oct4 and Rex1 respectively underwent demethylation by 23.04% (P < 0.01) and 6.02% (P > 0.05), and, reversely, Sox2, Nanog and Fgf4 respectively experienced remethylation by 0.84% (P > 0.05), 5.39% (P > 0.05) and 5.46% (P > 0.05) during MZT in NT embryos. Interestingly, the CpG 14 site of Sox2 was specifically methylated in both 8-cell and morula NT embryos. In addition, the development of blastocysts between IVF and NT embryos showed no significant difference. DNA methylation analysis showed that only Oct4 and Sox2 underwent the correct methylation reprogramming process, which may be responsible for the development of blastocysts of NT embryos to a certain extent. In conclusion, the five genes respectively experienced demethylation to different extents and incomplete DNA methylation reprogramming during bovine MZT in both IVF and NT embryos, suggesting that they may be used as indicators for bovine embryo developmental competence.

Ipr1 Gene Mediates RAW 264.7 Macrophage Cell Line Resistance to Mycobacterium bovis

Tuberculosis caused by Mycobacterium bovis (M. bovis) seriously affects efficiency of animal production with impacts on public health as well. Effective programmes of prevention and eradication of M. bovis infection therefore are urgently needed. Intracellular pathogen resistance gene 1 (Ipr1) is well known to mediate innate immunity to Mycobacterium tuberculosis (MTB), but there are no reports as to whether Ipr1 can enhance the phagocytic ability of macrophage against M. bovis. In this investigation, RAW 264.7 macrophage was transduced with lentiviral vector carrying Ipr1 (named Lenti‐Ipr1); transgenic cells were identified by RT‐PCR and western blotting. Transgenic positive cells (R‐Ipr1) were then infected with an M. bovis virulent strain, with non‐transduced cells used as control. When cell proliferation, viability and apoptosis of the two groups were investigated, it was found that infected RAW 264.7 died by necrosis whereas R‐Ipr1 underwent apoptosis. Furthermore, the numbers of intracellular bacteria in R‐Ipr1 were lower than those in control cells (P < 0.05). To identify the role of Ipr1, we measured the genes of Casp3, Mcl‐1 and NOS2A which associated with macrophage activation and apoptosis by real‐time quantitative PCR. The results demonstrated that Ipr1 gene expression can enhance anti‐M. bovis infection of macrophage. This establishes a basis for the future production of Ipr1‐transgenic cattle to strengthen the tuberculosis resistance.

Recombinant adenovirus-mediated human telomerase reverse transcriptase gene can stimulate cell proliferation and maintain primitive characteristics in bovine mammary gland epithelial cells.

The human telomerase reverse transcriptase (hTERT) gene has been used to stimulate the proliferation of most types of human cells. The present study was designed to evaluate the feasibility and efficiency of adenovirus‐mediated hTERT in the proliferation of bovine mammary gland epithelial cells (bMGEs). A plasmid and an adenovirus vector that carried hTERT, namely pEGFP‐ hTERT and Ad‐ hTERT, were constructed and transfected into bMGEs, respectively. In order to select the best strategy for stimulating cell proliferation, the adenovirus‐ and plasmid‐mediated hTERT were compared in terms of the positive cloning and transgenic efficiency. The results showed that only Ad‐ hTERT had high infection efficiency and produced a positive polyclone population (hTERT‐bMGEs). The characteristics of the hTERT‐bMGEs were investigated with further analysis by reverse transcription–polymerase chain reaction (RT–PCR), western blotting, proliferation assays, and flow cytometry, which showed that hTERT facilitated strong cell proliferation. Real‐time quantitative PCR showed a normal level of expression of beta‐casein, the caspase‐8 and c‐myc proto‐oncogene, and immunofluorescence demonstrated the properties of the epithelial cells. In conclusion, the adenovirus‐mediated hTERT gene could not only extend the cell lifespan, but also maintained the primary characteristics of the cells. It may be possible to extend the use of a wide variety of non‐human mammalian cells in this way. This study has provided additional insight into the mechanism of cell proliferation by demonstrating the lack of integration of the adenovirus‐mediated hTERT gene into the mammalian genome.

Histone and DNA methylation control by H3 serine 10/threonine 11 phosphorylation in the mouse zygote

AbstractBackground In the mammalian zygote, epigenetic reprogramming is a tightly controlled process of coordinated alterations of histone and DNA modifications. The parental genomes of the zygote show distinct patterns of histone H3 variants and distinct patterns of DNA and histone modifications. The molecular mechanisms linking histone variant-specific modifications and DNA methylation reprogramming during the first cell cycle remain to be clarified.ResultsHere, we show that the degree and distribution of H3K9me2 and of DNA modifications (5mC/5hmC) are influenced by the phosphorylation status of H3S10 and H3T11. The overexpression of the mutated histone variants H3.1 and 3.2 at either serine 10 or threonine 11 causes a decrease in H3K9me2 and 5mC and a concomitant increase in 5hmC in the maternal genome. Bisulphite sequencing results indicate an increase in hemimethylated CpG positions following H3.1T10A overexpression suggesting an impact of H3S10 and H3T11 phosphorylation on DNA methylation maintenance.ConclusionsOur data suggest a crosstalk between the cell-cycle-dependent control of S10 and T11 phosphorylation of histone variants H3.1 and H3.2 and the maintenance of the heterochromatic mark H3K9me2. This histone H3 “phospho-methylation switch” also influences the oxidative control of DNA methylation in the mouse zygote.

Construction of Ipr1 expression vector and development of cloned embryos in vitro.

The purpose of this study was to prepare intracellular pathogen resistance 1 (Ipr1) transgenic donor cells for somatic cell nuclear transfer (SCNT). Based on our current understanding of Ipr1, a macrophage special expression vector pSP-EGFP-Ipr1was constructed. Bovine fetal fibroblasts were transfected with pSP-EGFP-Ipr1. The green fluorescent protein (GFP)-expressing cells were selected and transferred into enucleated bovine oocytes. Then, the rates of oocyte cleavage and blastocyst formation of transgenic cells and non-transgenic cells were observed, respectively. The results showed that reconstructed embryos derived from transgenic cells could successfully develop into blastocysts, most of which were GFP-positive. This study may provide cloned embryos for the production of anti-tuberculosis transgenic animals.

cDNA cloning of goat DNA methyltransferase 1, screening of shRNA vectors and influences to development of nuclear transfer embryos.

Abstract This study was designed to clone cDNA of goat DNA methyltransferase 1 (DNMT1) gene, to screen an effective shRNA-producing vector targeting goat DNA methyltransferase 1 and to improve the developmental competence of goat nuclear transfer embryos by decreasing the DNMT1 expression in donor cells. In this study, PCR primers were designed against regions of high homology between bovine and sheep sequences and then used to amplify the larger portions of the coding regions. Next, 3 RNAi oligonucleotides were designed based on the cloned sequences and inserted into pRNAT-U6.1/Neo vector, acquiring 3 new vectors, respectively termed pRNAD1, pRNAD2 and pRNAD3. Then the positive cells were sorted by flow cytometry after transfection and detected by real-time PCR analysis and sodium bisulfite genomic sequencing. Finally, the developmental rates of nuclear transfer (NT) embryos generated using donor cells with and without the effective shRNA vector respectively, as well as in vitro fertilization (IVF) embryos were observed and recorded. The results showed that the coding regions of goat DNA methyltransferase 1 gene was successfully cloned (GenBank no. FJ617538). Furthermore, an effective interfering shRNA (pRNAD2) was obtained, with its interference effect being 47.88%. Finally, NT embryos with shRNA vector harbored better developmental competence during morula and blastocyst stage compared to controls ( P P > 0.05). In conclusion, goat DNA methyltransferase 1 gene cDNA was cloned and sequenced, an effective shRNA vector responsible for inhibiting DNA methyltransferase 1 expression was developed and the developmental competence of goat nuclear transfer morulae and blastcysts was significantly improved, which provided a feasible pathway for improving goat nuclear transfer embryo development competence by decreasing the methylation level in donor cells through RNAi-mediated manner.

DNA Methylation Reprogramming in Preimplantation Development

Shortly after fertilisation the embryonic cells pass through a transient state of totipotency followed by the formation of unipotent trophoblast and pluripotent embryonic stem cells, i.e. the first determined cell lineages which can be derived from the early blastocyst. The molecular processes leading to totipotency formation are initiated by molecules and enzymes provided by the egg cytoplasm. The first molecular changes that can be observed are extensive reconfigurations of the sperm chromatin accompanied by major changes in epigenetic marks of the DNA and chromatin. The epigenetic reprogramming starts in the paternal pronucleus of the zygote and eventually affects both parental chromosomes yielding strongly altered DNA and histone modifications at later developmental stages. In this chapter we will discuss the major molecular differences occurring during the first phase of epigenetic reprogramming with a focus on DNA modification dynamics in the mammalian zygote.