The first wave of transcription, called zygotic genome activation (ZGA), begins during the 2-cell stage in mouse preimplantation development and marks a vital transition from the maternal genetic to the embryonic genetic program. Utilizing DNA microarray data, we looked for genes that are expressed only during ZGA and found Zscan4, whose expression is restricted to late 2-cell stage embryos. Sequence analysis of genomic DNA and cDNA clones revealed nine paralogous genes tightly clustered in 0.85 Mb on mouse chromosome 7. Three genes are not transcribed and are thus considered pseudogenes. Among the six expressed genes named Zscan4a-Zscan4f, three - Zscan4c, Zscan4d, and Zscan4f - encode full-length ORFs with 506 amino acids. Zscan4d is a predominant transcript at the late 2-cell stage, whereas Zscan4c is a predominant transcript in embryonic stem (ES) cells. No transcripts of any Zscan4 genes are detected in any other cell types. Reduction of Zscan4 transcript levels by siRNAs delays the progression from the 2-cell to the 4-cell stage and produces blastocysts that fail to implant or proliferate in blastocyst outgrowth culture. Zscan4 thus seems to be essential for preimplantation development.

SUMMARY To examine transcription factor (TF) network(s), we created mouse ES cell lines, in each of which one of 50 TFs tagged with a FLAG moiety is inserted into a ubiquitously controllable tetracycline-repressible locus. Of the 50 TFs, Cdx2 provoked the most extensive transcriptome perturbation in ES cells, followed by Esx1, Sox9, Tcf3, Klf4, and Gata3. ChIP-Seq revealed that CDX2 binds to promoters of up-regulated target genes. By contrast, genes down-regulated by CDX2 did not show CDX2 binding, but were enriched with binding sites for POU5F1, SOX2, and NANOG. Genes with binding sites for these core TFs were also down-regulated by the induction of at least 15 other TFs, suggesting a common initial step for ES cell differentiation mediated by interference with the binding of core TFs to their target genes. These ES cell lines provide a fundamental resource to study biological networks in ES cells and mice.

No abstract

Analysis of gene expression changes during preimplantation development by quantitative reverse transcription-polymerase chain reaction (Q-PCR) requires appropriate internal standards. Ideally, such a gene should show a constant level of transcripts per embryo across all preimplantation stages from unfertilized eggs to blastocysts. By analysing the microarray-based gene expression profiles of preimplantation embryos, it was found that a conserved helix-loop-helix ubiquitous kinase gene (Chuk, also known as IkappaB kinase alpha, IKKalpha or IKK1) satisfied this criterion. To test the utility of this gene as an internal standard for Q-PCR, the expression levels of two known genes (Nalp5/Mater, Pou5f1/Oct3/Oct4) were normalized by Chuk and other housekeeping genes (Actb, Gapdh, Eef1a1, and H2afz) and demonstrated that the former was more consistent with the expression patterns obtained by a whole-mount in-situ hybridization than those reported previously with the latter. It is concluded that Chuk, unlike other commonly used normalization controls, is a reliable and suitable internal standard for measuring gene expression levels by Q-PCR in mouse oocytes and preimplantation embryos.

Cell-free fetal DNA in maternal plasma or serum is at present widely investigated as a source of fetal genetic material, both in studies of pregnancy-related disorders and in planning strategies for non-invasive prenatal diagnosis. Despite the number of trials already performed on the quantitation of fetal DNA, data about the amount of DNA at the beginning of pregnancy, in particular in the first trimester, remain limited. A new probe mapping on the deleted in azoospermia (DAZ) repetitive region of the Yq chromosome was designed for an early assessment of fetal DNA concentration in maternal serum. Among 57 pregnant women prospectively studied in their first trimester, fetal DNA was detected already by the 5th gestational week, with the analysis becoming reliable by the 8th week of gestation when a 100% accuracy in fetal sex determination was achieved. Moreover, in the three cases of pregnancy ending in fetal loss, the amount of fetal DNA apparently decreased before the abortion was diagnosed, whereas it consistently showed an increasing trend in normal pregnancies. Real-time PCR with the use of DAZ multilocus probe can efficiently quantitate free fetal DNA in the maternal serum at the beginning of pregnancy.

Radial deficiencies (RDs), defined as under/abnormal development or absence of any of the structures of the forearm, radial carpal bones and thumb, occur with a live birth incidence ranging from 1 out of 30,000 to 1 out 6,000 newborns and represent about one third/one fourth of all the congenital upper limb anomalies. About half of radial disorders have a mendelian cause and pattern of inheritance, whereas the remaining half appears sporadic with no known gene involved. In sporadic forms certain anomalies, such as thumb or radial hypoplasia, may occur either alone or in association with systemic conditions, like vertebral abnormalities or renal defects. All the cases with a mendelian inheritance are syndromic forms, which include cardiac defects (in Holt-Oram syndrome), bone marrow failure (in Fanconi anemia), platelet deficiency (in thrombocytopenia-absent-radius syndrome), ocular motility impairment (in Okihiro syndrome). The genetics of radial deficiencies is complex, characterized by genetic heterogeneity and high inter- and intra-familial clinical variability: this review will analyze the etiopathogenesis and the genotype/phenotype correlations of the main radial deficiency disorders in humans.

No abstract

National legislations represent one of the main factors influencing access to assisted reproduction treatment. The Italian situation in the last decade is an example of how the treatment of patients for preimplantation genetic diagnosis (PGD) was more dependent on regulators than on medical choices. This report analysed how the changes in Italian regulation affected the number of PGD referrals to this study centre, as well as their decision to opt for cross-border reproductive care (CBRC). The analysis showed that during the period in which PGD was actually not performed because of the restriction imposed by the Italian law on IVF (from 24 February 2004 to 7 May 2009) there was a significant decrease in the number of referrals asking for PGD (2.5% of total referrals) compared with the previous years (3.3%; P < 0.025) and following years when PGD was legalized (5.1%; P < 0.001). The number of couples opting for CBRC had an opposite trend, reaching a maximum when PGD was banned from Italian centres (55 couples), whereas after the readmission of PGD, only eight couples went abroad for treatment. Concomitantly, since May 2009, the proportion of couples performing a PGD cycle in this centre has constantly increased.