Generation of transgene-free mouse induced pluripotent stem cells using an excisable lentiviral system.

One goal of research using induced pluripotent stem cell (iPSC) is to generate patient-specific cells which can be used to obtain multiple types of differentiated cells as disease models. Minimally or non-integrating methods to deliver the reprogramming genes are considered to be the best but they may be inefficient. Lentiviral delivery is currently among the most efficient methods but it integrates transgenes into the genome, which may affect the behavior of the iPSC if integration occurs into an important locus. Here we designed a polycistronic lentiviral construct containing four pluripotency genes with an EGFP selection marker. The cassette was excisable with the Cre-loxP system making possible the removal of the integrated transgenes from the genome. Mouse embryonic fibroblasts were reprogrammed using this viral system, rapidly resulting in large number of iPSC colonies. Based on the lowest EGFP expression level, one parental line was chosen for excision. Introduction of the Cre recombinase resulted in transgene-free iPSC subclones. The effect of the transgenes was assessed by comparing the parental iPSC with two of its transgene-free subclones. Both excised and non-excised iPSCs expressed standard pluripotency markers. The subclones obtained after Cre recombination were capable of differentiation in vitro, in contrast to the parental, non-excised cells and formed germ-line competent chimeras in vivo.

Quality analysis of buffalo blastocysts derived from oocytes vitrified before or after enucleation and reconstructed with somatic cell nuclei.

We investigated the potential of vitrified-warmed buffalo oocytes to develop to blastocysts after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT). In vitro-matured oocytes before and after enucleation (M-II oocytes and enucleated oocytes, respectively) were put in 7.5% DMSO and 7.5% ethylene glycol (EG) for 4, 7 and 10 min, and then vitrified (Cryotop device) after 1-min equilibration in 15% DMSO, 15% EG and 0.5M sucrose. Following 4-, 7- and 10-min exposure, proportions of the post-warm oocytes with a normal vitelline membrane were similar (66-71% in M-II oocytes and 69-71% in enucleated oocytes). However, 18-20% of the normal M-II oocytes had no detectable first polar body in their perivitelline space (no potential for subsequent enucleation). When the post-warm M-II oocytes were treated for PA by 7% ethanol, 10 microg/mL cycloheximide and 1.25 microg/mL cytochalasin-D, parthenogenetic development into Day-7 blastocysts occurred in 10-13% of cultured oocytes, lower (P<0.05) than fresh (control) oocytes (24%). In the absence of the cooling and warming, blastocyst rates in the 4-min exposure group (22%), but not in the 7-min and 10-min exposure groups (14-15%), were similar to that in the fresh group (23%). The total cell number (group average 117-132 cells) and the ICM ratio (22-24%) of the PA blastocysts derived from vitrified M-II oocytes were comparable with fresh oocytes (127 cells and 25%). After SCNT (with fibroblast cells and vitrified-warmed oocytes), blastocyst rates were similar for the three exposure periods for M-II oocytes (8-10%) and enucleated oocytes (7-9%), but were lower (P<0.05) than in the fresh group (15%). The total cell number of the SCNT blastocysts derived from vitrified M-II and enucleated oocytes (80-90 and 82-101 cells) was smaller (P<0.05) than from fresh oocytes (135 cells); the ICM ratio of blastocysts derived from the M-II and enucleated oocytes after vitrification in 7- or 10-min exposure groups (20-22%) was not different (P>0.05) from fresh control oocytes (24%) or those in 4-min exposure group (M-II 23%, enucleated 24%). Thus, SCNT of swamp buffalo oocytes following vitrification before or after enucleation resulted in blastocysts with a slightly decreased cell number.

Epigenetic characteristics of cloned and in vitro-fertilized swamp buffalo (Bubalus bubalis) embryos.

Swamp buffalos are becoming endangered due to reproductive inefficiencies. This is of concern because many countries depend heavily on their products. Somatic cell nuclear transfer (SCNT) is a potential strategy for preserving endangered species. To date, SCNT in swamp buffalo has succeeded in the creation of blastocyst embryos. However, development to term of SCNT swamp buffalos is extremely limited, and only 1 live birth has been reported. An abnormal epigenetic mechanism is suspected to be the cause of developmental failure, as is also seen in other species. The DNA methylation and histone acetylation are key players in epigenetic modification and display marked variability during embryonic preimplantation development. Knowledge of epigenetic modifications will aid in solving the developmental problems of SCNT embryos and improving reproductive technology in the swamp buffalo. The objective of this study was to determine the relationship between preimplantation embryonic development and 2 epigenetic patterns, global DNA methylation and histone acetylation, in SCNT and in vitro-fertilized (IVF) swamp buffalo embryos. In addition, we examined the correlations between those 2 mechanisms in the SCNT and IVF swamp buffalo embryos throughout the developmental stages using double immunostaining and quantification of the emission intensities using confocal microscopy. We discovered an aberrant methylation pattern in early preimplantation-stage swamp buffalo SCNT embryos. In addition, greater variability in the DNA methylation levels among nuclei within SCNT embryos was discovered. Hyperacetylation was also observed in SCNT embryos compared with IVF embryos at the 4- and 8-cell stages (P < 0.05). Dynamic changes and interplay between these 2 epigenetic mechanisms could be crucial for embryonic development during the early preimplantation period. The aberrancies uncovered here may contribute to the low efficiency of SCNT.

Anomalous mRNA levels of chromatin remodeling genes in swamp buffalo (Bubalus bubalis) cloned embryos.

The swamp buffalo (Bubalus bubalis) is a multi-purpose animal in agriculture that is challenged by extinction due to low reproductive efficiency. Nuclear transfer (NT) has been used to preserve special breeds of buffalo, as well as to increase the number of animals. However, cloned buffalo embryos have impaired development, as in other species. To understand the chromatin remodeling activities in cloned embryos and to improve NT technology, we examined the expression profiles of five genes involved in DNA and histone modifications, DNMT1, DNMT3A, DNMT3B, HAT1 and HDAC1, in single swamp buffalo metaphase II oocytes, NT and in vitro fertilized (IVF) embryos from the two-cell to the blastocyst stage, by quantitative real time RT-PCR. We observed similar expression dynamics for all genes studied in the NT and IVF embryos: relatively constant levels of expression for all genes were found from the MII oocyte up to the eight-cell stage; the levels of mRNA for HAT1 and DNMT3B continued to be stably expressed up to the blastocyst stage; while dramatic increases were seen for DNMT3A and HDAC1. Alternatively, the levels of DNMT1 started to decrease at the eight-cell stage. Despite the similarity in the dynamics of gene expression, dramatic differences in the relative levels of these genes between NT and IVF embryos were observed. The expression levels of all DNA modifying genes were higher in the NT embryos than in the IVF embryos at the eight-cell and blastocyst stages. The genes HDAC1 and HAT1 were also expressed significantly higher at the blastocyst stage in the NT embryos. Our results suggested differences in chromatin remodeling between NT and IVF embryos and that lower levels of DNA passive demethylation and higher levels of DNA de novo methylation occurred in the NT embryos. These observations are novel in the species of buffalo, and may be associated with developmental failure of cloned buffalo embryos due to the transcriptional repression effect of most genes studied here.