RICERCA ITALIANA     

Research program

Embryonic stem-like cells obtained from fibroblasts cultured in presence of mouse embryonic stem cell extracts

University Co-ordinator

Università degli Studi di PARMA - MEDICINA SPERIMENTALE - PARMA(PR)

Research Unit Leader

Maurizio ZUCCOTTI

Description

At each time interval (every 48 hr) samples prepared by Research Unit N. 1, will be handled as for the analysis of gene expression and DNA methylation. These experiments will be carried out on ES cells (control) and on fibroblasts, before (control) and after they have been cultured in ES extracts.


GENE EXPRESSION
Analysis of the onset and profile of abundance of genes that mark the reprogramming process.
- ES cells specific genes: Oct-4, Rex-1 and Nanog.
- Fibroblasts specific genes: Col1a1, Col1a2 e FSP1/S100a4.
Gene expression will be analysed at specific time intervals (i.e., every 48 hr) following exposure of fibroblasts to the ES extract.

RNA isolation and cDNA synthesis
RNA will be isolated and retrotranscribed following Gentile et al. (2004). pAW109 RNA (Applera) will be added to each sample as an internal control for efficiency of the RT-PCR (Retro Transcriptase Polymerase Chain Reaction) and it will be used as a standard to enable the comparison with the relative abundance of transcripts of the genes under study.

PCR
For each gene we will set up a triplex PCR for the co-amplification, in the first PCR, of three gene sequences: the cDNA sequence of the gene under study, the control cDNA used for quantitation (i.e., pAW109) and a control endogenous gene (i.e., Hprt).

Quantitation of PCR products
Following electrophoresis, the products will be visualised under short-wave length UV on Bio-Rad Gel Doc system and densitometric analysis will be performed with the Bio-Rad Quantity-One software. In addition, following the retro transcription step, samples will also be analysed with the Taqman procedure (Applera) which will allow a real-time PCR analysis.


DNA METHYLATION OF THE ENTIRE GENOME AND OF SPECIFIC GENES
To analyse DNA methylation pattern in toto, we will make use of an anti-5-methylcytosine antibody. We will also analyse the CpG methylation pattern of the promoter and 5' region of Oct-4, Rex-1 and Nanog, making use of the methylation-sensitive PCR and the bisulfite method.

Analysis in situ
Cells will be rinsed from culture medium, centrifuged, fixed with ice-cold methanol:acetic acid (3:1, v:v) and layed onto a slide. Slides will be incubated in PBS supplemented with 3% bovine serum albumine (BSA) for 20 min at 37º C in order to block aspecific binding sites, then they will be incubated with anti-5-methylcytosine FITC labeled in PBS, 1% BSA for 60 min at 37º C. Following 3 washes in PBS, nuclei will be stained with Hoechst 33258 (0.2µg/ml) for 5 min, washed in PBS and mounted in anti-fading agent (DABCO). To exclude anti-5-methylcytosine differential staining, due to differential accessibility of DNA, nuclei will be stained with anti-DNA antibody.

Analysis extra situm
Following DNA extraction, methylation of CpG dinucleotides will be studied by both the restriction enzyme-sensitive PCR assay and by the bisulfite genomic sequencing method.

Restriction enzyme-sensitive PCR
30 U of methylation sensitive restriction enzymes will be added to the DNA sample and incubated for 90 min at 37º C followed by heat inactivation of the restriction enzyme at 95º C for 15 min. Twenty-three µl of PCR mixture will be added to each DNA sample. For each gene, primers will be designed such that the sequence amplified will include one or more informative CpG sites (Zuccotti et al., 1993). Samples will be electrophoresed on agarose gel.

Bisulfite method
DNA samples will be denatured in 3M NaOH, and sodium metabisulfite (2M final concentration), hydroquinone (0.5 mM final concentration), 2 µg E. coli tRNA will be added and the mixture incubated for 4 hr at 50º C. DNA samples will be desalted (using the wizard DNA clean-up system, Promega), eluted in dH2O, desulfonated (in 3M NaOH) and finally precipitated in ethanol. PCR of bisulfite-treated DNA and control untreated-DNA will be run with the appropriate primers in a standard PCR reaction mixture. Amplification products will be sequenced.


BIBLIOGRAPHIC REFERENCES

Gentile L., Monti M., Sebastiano V., Merico V., Garagna S., Redi C.A., Zuccotti M.: Single-cell quantitative RT-PCR analysis of Cpt-1b and Cpt-2 gene expression in mouse antral oocytes and in preimplantation embryos. Cytogenet Genome Res 105: 215-221, 2004.

Zuccotti M., Grant M., Monk M.: Polymerase chain reaction for the detection of methylation of a specific CpG site in the G6pd gene of mouse embryos. In: Methods in Enzymology Vol. 225: "A guide to techniques in mouse development". Wassarman P. and DePamphilis M.L. (eds.). Academic Press, San Diego, pp. 557-567, 1993.