A short G1 phase is a characteristic feature of mouse embryonic stem cells (ESCs). To determine if there is a causal relationship between G1 phase restriction and pluripotency, we made use of the Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) reporter system to FACS-sort ESCs in the different cell cycle phases. Hence, the G1 phase cells appeared to be more susceptible to differentiation, particularly when ESCs self-renewed in the naïve state of pluripotency. Transitions from ground to naïve, then from naïve to primed states of pluripotency were associated with increased durations of the G1 phase, and cyclin E-mediated alteration of the G1/S transition altered the balance between self-renewal and differentiation. LIF withdrawal resulted in a lengthening of the G1 phase in naïve ESCs, which occurred prior to the appearance of early lineage-specific markers, and could be reversed upon LIF supplementation. We concluded that the short G1 phase observed in murine ESCs was a determinant of naïve pluripotency and was partially under the control of LIF signaling.

Leukemia inhibitory factor (LIF)/STAT3 signalling is a hallmark of naive pluripotency in rodent pluripotent stem cells (PSCs), whereas fibroblast growth factor (FGF)-2 and activin/nodal signalling is required to sustain self-renewal of human PSCs in a condition referred to as the primed state. It is unknown why LIF/STAT3 signalling alone fails to sustain pluripotency in human PSCs. Here we show that the forced expression of the hormone-dependent STAT3-ER (ER, ligand-binding domain of the human oestrogen receptor) in combination with 2i/LIF and tamoxifen allows human PSCs to escape from the primed state and enter a state characterized by the activation of STAT3 target genes and long-term self-renewal in FGF2- and feeder-free conditions. These cells acquire growth properties, a gene expression profile and an epigenetic landscape closer to those described in mouse naive PSCs. Together, these results show that temporarily increasing STAT3 activity is sufficient to reprogramme human PSCs to naive-like pluripotent cells.

The development of transgenic technologies in monkeys is important for creating valuable animal models of human physiology so that the etiology of diseases can be studied and potential therapies for their amelioration may be developed. However, the efficiency of producing transgenic primate animals is presently very low, and there are few reports of success. We have developed an improved methodology for the production of transgenic rhesus monkeys, making use of a simian immunodeficiency virus (SIV)-based vector that encodes EGFP and a protocol for infection of early-cleavage–stage embryos. We show that infection does not alter embryo development. Moreover, the timing of infection, either before or during embryonic genome activation, has no observable effect on the level and stability of transgene expression. Of 70 embryos injected with concentrated virus at the one- to two-cell stage or the four- to eight-cell stage and showing fluorescence, 30 were transferred to surrogate mothers. One transgenic fetus was obtained from a fraternal triple pregnancy. Four infant monkeys were produced from four singleton pregnancies, of which two expressed EGFP throughout the whole body. These results demonstrate the usefulness of SIV-based lentiviral vectors for the generation of transgenic monkeys and improve the efficiency of transgenic technology in nonhuman primates.

MHC genes in the chicken are arranged into two genetically independent clusters located on the same chromosome. These are the classical B system and restriction fragment pattern-Y (Rfp-Y), a second cluster of MHC genes identified recently through DNA hybridization. Because small numbers of MHC class I and class II genes are present in both B and Rfp-Y, the two clusters might be the result of duplication of an entire chromosomal segment. We subcloned, sequenced, and analyzed the expression of two class I loci mapping to Rfp-Y to determine whether Rfp-Y should be considered either as a second, classical MHC or as a region containing specialized MHC-like genes, such as class Ib genes. The Rfp-Y genes are highly similar to each other (93%) and to classical class Ia genes (73% with chicken B class I; 49% with HLA-A). One locus is disrupted and unexpressed. The other, YFV, is widely transcribed and polymorphic. Mature YFV protein associated with β₂m arrives on the surface of chicken B (RP9) lymphoma cells expressing YFV as an epitope-tagged transgene. Substitutions in the YFV Ag-binding region (ABR) occur at four of the eight highly conserved residues that are essential for binding of peptide-Ag in the class Ia molecules. Therefore, it is unlikely that Ag is bound in the YFV ABR in the manner typical of class Ia molecules. This ABR specialization indicates that even though YFV is polymorphic and widely transcribed, it is, in fact, a class Ib gene, and Rfp-Y is a region containing MHC genes of specialized function.