In mice, gene targeting by homologous recombination continues to play an essential role in the understanding of functional genomics. This strategy allows precise location of the site of transgene integration and is most commonly used to ablate gene expression ("knockout"), or to introduce mutant or modified alleles at the locus of interest ("knock-in"). The efficacy of producing live, transgenic mice challenges our understanding of this complex process, and of the factors which influence germline competence of embryonic stem cell lines. Increasingly, evidence indicates that culture conditions and in vitro manipulation can affect the germline-competence of Embryonic Stem cell (ES cell) lines by accumulation of chromosome abnormalities and/or epigenetic alterations of the ES cell genome. The effectiveness of ES cell derivation is greatly straindependent and it may also influence the germline transmission capability. Recent technical improvements in the production of germline chimeras have been focused on means of generating ES cells lines with a higher germline potential. There are a number of options for generating chimeras from ES cells (ES chimera mice); however, each method has its advantages and disadvantages. Recent developments in induced pluripotent stem (iPS) cell technology have opened new avenues for generation of animals from genetically modified somatic cells by means of chimera technologies. The aim of this review is to give a brief account of how the factors mentioned above are influencing the germline transmission capacity and the developmental potential of mouse pluripotent stem cell lines. The most recent methods for generating specifically ES and iPS chimera mice, including the advantages and disadvantages of each method are also discussed.

The efficiency of various assisted reproductive techniques can be improved by preconditioning the gametes and embryos with sublethal hydrostatic pressure treatment. However, the underlying molecular mechanism responsible for this protective effect remains unknown and requires further investigation. Here, we studied the effect of optimised hydrostatic pressure treatment on the global gene expression of mouse oocytes after embryonic genome activation. Based on a gene expression microarray analysis, a significant effect of treatment was observed in 4-cell embryos derived from treated oocytes, revealing a transcriptional footprint of hydrostatic pressure-affected genes. Functional analysis identified numerous genes involved in protein synthesis that were downregulated in 4-cell embryos in response to hydrostatic pressure treatment, suggesting that regulation of translation has a major role in optimised hydrostatic pressure-induced stress tolerance. We present a comprehensive microarray analysis and further delineate a potential mechanism responsible for the protective effect of hydrostatic pressure treatment.

Induced pluripotent stem (iPS) cell technology allows the reprogramming of somatic cells to a pluripotent state; however, it requires viral gene transduction and permanent existence of the exogenous genes in the genome, which is a potential risk for abnormalities in the derived iPS cells. Recently, there was report that iPS cells have been made with piggyBack transposon. Here, we first reported that nonviral transfection of a Sleeping Beauty transposon, which comprises c-Myc, Klf-4, Oct3/4 (Pou5f1), and Sox-2, can reprogram mouse fibroblasts from 3 different genetic backgrounds: ICR (outbred), C57BL/6 (inbred), and F1 hybrid (C57BL/6 × DBA/2J), with parallel robust expression of all exogenous (c-Myc, Klf-4, Oct3/4, and Sox-2) and endogenous (e.g. Nanog) pluripotency genes. The iPS cells were cultured under standard conditions with promotion of differentiate by withdrawal of leukemia inhibitory factor. We chose 6 cloned of each line that exhibited characteristics typical for undifferentiated embryonic stem (ES) cell: ES-cell-like morphology, alkaline phosphatase positivity, and gene expression pattern [quantitative real-time PCR and immunofluorescence of ES cell markers (e.g. Oct-4, SSEA1, Nanog]. Furthermore, cells were able to form embryoid bodies and beat rhythmically and expressed cardiac markers assayed by immunofluorescence (e.g. cardiac Troponin T, desmin). In vivo testing of iPS cell lines for their developmental potential (diploid and tetraploid embryo complementation assay) is currently underway. The iPS cell lines generated from the ICR strain appeared the earliest in time (ICR-d11, F1 day-2 and Bl6-d12), with higher efficiency than colonies from the other 2 backgrounds. The differentiation potential of the iPS lines derived from the 3 genetic backgrounds was similar. Interestingly, the ICR-iPS lines had higher differentiation potential than did the ICR-ES cell lines: the rate of embryoid bodies forming rhythmically beating cardiomyocytes was 4% in ICR-ES and 79% in ICR-iPS cells, respectively. Our results suggest that the iPS technology provide a new tool to generate pluripotent stem cells from genetic backgrounds where good-quality ES cell generation is difficult. These studies provide new insights into virus-free iPS technology and contribute to defining future cell-based therapies, drug screening methods, and production of transgenic animals with genetically modified iPS cells. This study was financed by EU FP6 (CLONET, MRTN-CT-2006-035468), EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; InduStem, PIAP-GA-2008-230675; PluriSys, HEALTH-2007-B-223485); NKTH-OTKA-EU FP7-HUMAN-2009-MB08-C 80205, and NKTH/KPI (Jedlik NKFP_07_1-ES2HEART-HU OM-00202-2007).

Sublethal stress treatment [namely hydrostatic pressure (HP)] has been reported to improve the cryotolerance and developmental competence of mammalian gametes and embryos at cryopreservation, in vitro maturation, parthenogenetic activation and somatic cell NT (Pribenszky and Vajta 2011 Reprod. Fertil. Dev. 23, 48–55). In our previous study mouse oocytes were HP stress treated and then vitrified, warmed, fertilized by intracytoplasmic sperm injection (ICSI), cultured and transferred. Blastocyst rate, inner cell mass cell number and birth rate were significantly increased in the treated groups (Pribenszky et al. 2010 Fertil. Steril. 94, S32). In our present study we implemented gene expression microarray experiments to analyse the transcriptome of HP-treated eggs and 4-cell stage embryos developed from these oocytes. At first, 4 pools of 50 mouse oocytes were treated with 20 MPa for 60 min at 37°C in a programmable HP machine (Cryo-Innovation Inc., Budapest, Hungary). Second, mouse oocytes were treated with HP likewise, fertilized by ICSI and cultured to 4-cell stage. The controls were handled identically with no HP treatment. After ICSI, 78% of the treated and 82% of the control oocytes survived the procedure, of which 76 and 65% developed to 4-cell stage; furthermore 3–3 pools of 30 to 44 and 24 to 36 embryos were used for RNA isolation, respectively. The labelled cRNA target generated from 240 pg of total RNA was hybridized to 4 × 44 K whole mouse genome chips (Agilent Technologies, Palo Alto, CA, USA). All the microarray data have been deposited in the NCBI Gene Expression Omnibus database (accession GSE28443). The results were validated using RT-qPCR by analysing 9 genes per experiment. The GeneSpring GX 11 analysis of the normalized data showed no change in the global gene expression profile of the oocytes after the HP treatment; however, in the 4-cell embryo study, the analysis identified 676 significantly changed genes, confirmed by the similar expression of 8 genes by RT-qPCR. To characterise the affected pathways among these genes, we used the functional annotation tools of DAVID Bioinformatics Resources (Huang et al. 2009 Nat. Protoc. 4, 44–57). The cluster with the most outstanding gene ontology terms contained the ribosome, structural constituent of ribosome, ribonucleoprotein complex, translation and structural molecule activity (P < 0.001). Among the 2 directly ribosome-related categories, there were 35 and 40 down-regulated genes along with only 2-2 up-regulated genes. These results showed that principally the protein synthesis machinery is affected by the HP stress, with a robust down-regulation. In conclusion, this study suggests that ribosomal processes play a central role in the HP-treated and fertilized oocytes during pre-implantation development. Stress effects in the transcriptional processes are visible after the embryonic genome activation showing down-regulation in the most energy-consuming processes. How these processes support higher blastocyst rate, cell number and birth rate needs further elucidation. Supported by OM-00069/2008, EGG_Care; EU FP7 (EpiHealth FP7-HEALTH-2011-278418).

The progress of molecular genetics generated thousands of new transgenic strains of mice which also requires their economic and safe maintenance in the form of genetic banks. Sperm freezing would be one of the easiest options; however, cryosensitivity of sperm in mice strains is prone to variation. In this study, we examined the efficiency of laser-assisted zona drilling in vitro fertilization (ZD-IVF) v. intracytoplasmic sperm injection (ICSI) for attempting to recover two transgenic (UBI-GFP/BL6 and B6;129P2- Hvcn1) and one mutant (C57BL/6J-Tyrc-2J) lines. The sperm was frozen with 18% raffinose and 3% skim milk (Nakagata 2000 Mamm Genome 11, 572–576). Data of the replicates was analysed by chi-square method. The motility rates after thawing of cryopreserved sperm were 10% for UBI-GFP/BL6, 30% C57BL/6J-Tyrc-2J and 50% for B6;129P2-Hvcn1 strains. Regular IVF attempts in the UBI-GFP/BL6 and the mutant strain resulted in very few embryos and no pups (data not shown). Following ZD-IVF, the 2-cell stage rates were 7/60 (12%) for UBI-GFP/BL6, 18/60 (30%) for C57BL/6J-Tyrc-2J, and 34/60 (56%) for B6;129P2- Hvcn1. After ICSI, 66–74% of the oocytes survived the procedure and their development to 2-cells stage were 12/20 (60%) (UBI-GFP/BL6), 25/39 (64%) (C57BL/6J-Tyrc-2J) and 26/37 (70%) (B6;129P2-Hvcn1). The number of 2-cell stage embryos produced by ICSI was significantly (P < 0.05) increased compared with those produced following ZD-IVF in case of UBI-GFP/BL6 and C57BL/6J-Tyrc-2J strains. The 2-cell stage embryos were transferred into recipients and the newborn rates from ZD-IVF v. ICSI embryos were 0% v. 17% (UBI-GFP/BL6), 28% v. 36% (C57BL/6J-Tyrc-2J) and 12% v. 12% (B6;129P2- Hvcn1), respectively; none of them were significantly different. In conclusion, when using cryopreserved sperm, the post-thaw motility is an important indicator for the selection of the rederivation method of cryopreserved transgenic mouse strains; while ZD-IVF, an easier method to perform, is suitable for the higher motility samples, ICSI could be strongly recommended for those showing low motility. This work was financed by EU FP6: CLONET (MRTN-CT-2006-035468), TEAMOHOLIC (MEXT-CT-2003-509582); EU FP7: RESOLVE (FP7-HEALTH-F4-2008-202047), RabPStem (PERG07-GA-2010-268422), and NKFP_07_1-ES2HEART-HU (OM-00202-2007).