Biomedical subjects

Markus Gilles

Publications and source records attributed to Markus Gilles.

4 recordsLinked to original sources

PubMed2006

Targeted suppression of E-cadherin gene expression in bovine preimplantation embryo by RNA interference technology using double-stranded RNA.

RNA interference (RNAi) has become acknowledged as an effective and useful tool to study gene function in diverse groups of cells. We aimed to suppress the expression of the E-cadherin gene during in vitro development of bovine preimplantation embryos using RNAi approach. In this experiment the effect of microinjection of E-cadherin and Oct-4 (as control) double-stranded (ds) RNA on the mRNA and protein expression level of the target E-cadherin gene was investigated. For this, a 496 bp long bovine E-cadherin and 341 bp long Oct-4 dsRNA sample were prepared using in vitro transcription. In vitro produced bovine zygotes were categorized into four treatment groups including those injected with E-cadherin dsRNA, Oct-4 dsRNA, RNase-free water, and uninjected controls. While the injection of E-cadherin dsRNA resulted in the reduction of E-cadherin mRNA and protein levels at the morula and blastocyst stage, the transcript and protein product remained unaffected in the Oct-4 dsRNA, water injected and uninjected control groups. The relative abundance of E-cadherin mRNA in the E-cadherin dsRNA injected morula stage embryos was reduced by 80% compared to the control group (P < 0.05). The Western blot analysis also showed a significant decrease in the E-cadherin protein (119 kDa) in E-cadherin dsRNA injected embryos compared to the other three groups. Microinjection of E-cadherin dsRNA has resulted only 22% blastocyst rate compared to 38%-40% in water injected and uninjected controls. In conclusion, our results indicated the suppression of E-cadherin mRNA and protein has resulted in lower blastocyst rate and the RNAi technology is a promising approach to study the function of genes in early bovine embryogenesis.

Animals↗

PubMed2006

Bovine blastocyst diameter as a morphological tool to predict embryo cell counts, embryo sex, hatching ability and developmental characteristics after transfer to recipients.

The aim of the present study was to explore whether the blastocyst diameter and the zona thickness at 168 h after fertilisation are useful parameters to predict quality and viability of bovine in-vitro-produced (IVP)-embryos. Although significant (P <l 0.05), the blastocyst diameter at 168 h correlated only poorly with the total number of cells (R(2) = 0.13) and with the number of trophectoderm (TE) cells (R(2) = 0.17). Hatched blastocysts (n = 66) at 216 h had a significantly greater mean diameter at 168 h (194.8 +/- 16.8 microm) compared with either blastocysts that had started but not finished hatching at 216 h (n = 26, 178.4 +/- 16.7 microm) or failed to commence hatching (n = 136, 162.7 +/- 12.9 microm). Transfer of 101 IVP blastocysts to synchronised recipients resulted in the birth of 38 calves (38%). There were significantly more bull calves born than cow calves (P < 0.05), but this was not correlated with blastocyst diameter or zona thickness at 168 h. There was also no correlation between the diameter of blastocysts or the zona thickness at 168 h and parameters of subsequent developmental characteristics, including rates of pregnancy, resorptions and abortions, pregnancy duration, delivery to term and birthweight. Overall, the present results indicate that the blastocyst diameter and the zona thickness at 168 h are good predictors for subsequent hatching ability in vitro, but not for the number of TE cells, inner cell mass cells or total cells and neither for subsequent developmental characteristics after transfer to recipients.

Animals↗

PubMed2004

Quantitative expression analysis of blastocyst-derived gene transcripts in preimplantation developmental stages of in vitro-produced bovine embryos using real-time polymerase chain reaction technology.

The main objective of the present study was to analyse the quantitative expression pattern of genes from a subtracted blastocyst transcriptome throughout the preimplantation developmental stages of in vitro-produced bovine oocytes and embryos. For this purpose, Day 5 morula (M) cDNAs were subtracted from Day 7 blastocyst (B) cDNAs (B-M) and used to establish a B-M subtracted cDNA library, as reported previously. From the total generated clones, 19 were analysed quantitatively. The mRNA samples isolated from pools of immature oocytes (n = 150), mature oocytes (n = 150) and two-cell (n = 80), four-cell (n = 40), eight-cell (n = 20), morula (n = 6) and blastocyst (n = 3) embryos were reverse transcribed and subjected to real-time polymerase chain reaction (PCR) using sequence-specific primers and SYBR green as the DNA dye. A relative standard curve method was used to analyse the real-time data taking the morula stage as a calibrator. Applying suppression subtractive hybridisation (SSH), a total of 71 clones, which represent 33 different expressed sequence tags, were generated and available for analysis. Most transcripts were analysed for the first time in bovine embryogenesis. The real-time PCR has validated the results of SSH positively for 84% (16/19) of transcripts, whereas 16% (3/19) showed deviation in the expression pattern from the one seen during SSH. Several transcript-specific expression patterns were observed for genes that play decisive roles in bovine embryogenesis. In addition to identification, accurately quantifying the expression profiles of transcripts during development will pave the way towards understanding the molecular mechanisms of embryogenesis and their potential role in early embryo development. Most importantly, the present study has contributed to the enrichment of bovine embryo gene collection by generating new transcripts involved in bovine embryo development.

Animals↗

PubMed2003

Identification and quantification of differentially expressed transcripts in in vitro-produced bovine preimplantation stage embryos.

In this study, we used mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) to analyze the mRNA expression patterns in in vitro-produced bovine 8-cell, 16-cell, morula, and blastocyst stage embryos and isolate differentially expressed amplicons. Moreover, we have used a fluorescence monitored real time quantitative PCR to quantify and analyze the expression patterns of the target differentially expressed transcripts through out the preimplantation stages from oocytes to blastocyst. For this, total RNA isolated from bovine 8-cell (n = 188), 16-cell (n = 94), morula (n = 35), and blastocyst (n = 15) were reverse transcribed and subjected to DDRT-PCR. Target differentially expressed transcripts were quantified by real time quantitative PCR. The cDNA banding pattern analysis revealed that large number of cDNA bands were conserved at 8-cell and blastocyst stage with a slight decrease at the morula stage. A total of 16 amplicons were cloned and sequenced. All expressed sequence tags (ESTs), except 1C19, showed sequence similarity with known genes or ESTs in GenBank. Sixty-two percent (10/16) of cDNA bands representing differentially expressed genes originated from 8-cell stage and the rest derived from the 16-cell, morula, or blastocyst stage. The quantitative PCR analysis has validated the expression patterns of 75% (12/16) of our transcripts to be in agreement with the results of DDRT-PCR. However, the quantitative PCR results of four transcripts showed a deviation from the pattern seen in DDRT-PCR. In conclusion, we have successfully applied mRNA DDRT-PCR to identify and isolate stage-specific expressed genes in bovine preimplantation embryos. In addition to validating the results of DDRT-PCR, quantitative real time PCR provides quantitative data on the expression of target genes.

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