Production of inbred offspring by intracytoplasmic sperm injection of oocytes from juvenile female mice.

The aim of the present study was to determine whether the use of oocytes from juvenile female mice would improve the efficiency of intracytoplasmic sperm injection (ICSI). In the present study, 15 adult and 14 juvenile C57BL6/J female mice were superovulated, with 17.8 oocytes per mouse harvested from adults, significantly lower than the 40.2 harvested from juveniles (P<0.01). Sixty and 233 oocytes were harvested from C57BL/6J adult and juvenile mice respectively, activated in 10mM SrCl2+5µgmL-1 cytochalasin B for 5-6h and cultured in potassium simplex optimisation medium (KSOM) for 3.5 days, with no differences in morula and blastocyst rates between groups (91.7% vs 96.6%; P>0.05). Twelve hours after injection of human chorionic gonadotrophin, oocytes were harvested from C57BL/6J juvenile mice into KSOM, randomly divided into groups and activated with the same method mentioned above at 0, 2, 4 or 6h and then cultured in KSOM for 3.5 days. There was no significant difference in morula and blastocyst rates among the different groups (P>0.05). Oocytes from juvenile mice activated in 10mM SrCl2 for 2h were subjected to ICSI and the rates of pronuclear formation and Day 1 cleavage were significantly improved compared with the control group (P<0.01). ICSI combined with activation of oocytes from inbred mouse strains (C57BL/6J, C57BL/6N and 129Svev) successfully produced pups. The fertility of some these mice resulting from ICSI was tested, and the animals proved fertile. In conclusion, superovulated juvenile mice can yield more useable oocytes than adult mice, but additional activation is essential for full development of ICSI oocytes harvested from juvenile inbred mice.

Long-range chromatin regulatory interactions in vivo.

Communication between distal chromosomal elements is essential for control of many nuclear processes. For example, genes in higher eukaryotes often require distant enhancer sequences for high-level expression. The mechanisms proposed for long-range enhancer action fall into two basic categories. Non-contact models propose that enhancers act at a distance to create a favorable environment for gene transcription, or act as entry sites or nucleation points for factors that ultimately communicate with the gene. Contact models propose that communication occurs through direct interaction between the distant enhancer and the gene by various mechanisms that 'loop out' the intervening sequences. Although much attention has focused on contact models, the existence and nature of long-range interactions is still controversial and speculative, as there is no direct evidence that distant sequences physically interact in vivo. Here, we report the development of a widely applicable in situ technique to tag and recover chromatin in the immediate vicinity of an actively transcribed gene. We show that the classical enhancer element, HS2 of the prototypical locus control region (LCR) of the beta-globin gene cluster, is in close physical proximity to an actively transcribed HBB (beta-globin) gene located over 50 kb away in vivo, suggesting a direct regulatory interaction. The results give unprecedented insight into the in vivo structure of the LCR-gene interface and provide the first direct evidence of long-range enhancer communication.

CAPRI and RASAL impose different modes of information processing on Ras due to contrasting temporal filtering of Ca2+.

The versatility of Ca2+ as a second messenger lies in the complex manner in which Ca2+ signals are generated. How information contained within the Ca2+ code is interpreted underlies cell function. Recently, we identified CAPRI and RASAL as related Ca2+-triggered Ras GTPase-activating proteins. RASAL tracks agonist-stimulated Ca2+ oscillations by repetitively associating with the plasma membrane, yet CAPRI displays a long-lasting Ca2+-triggered translocation that is refractory to cytosolic Ca2+ oscillations. CAPRI behavior is Ca2+- and C2 domain-dependent but sustained recruitment is predominantly Ca2+ independent, necessitating integration of Ca2+ by the C2 domains with agonist-evoked plasma membrane interaction sites for the pleckstrin homology domain. Using an assay to monitor Ras activity in real time, we correlate the spatial and temporal translocation of CAPRI with the deactivation of H-Ras. CAPRI seems to low-pass filter the Ca2+ signal, converting different intensities of stimulation into different durations of Ras activity in contrast to the preservation of Ca2+ frequency information by RASAL, suggesting sophisticated modes of Ca2+-regulated Ras deactivation.

An intergenic non-coding RNA promoter required for histone modifications in the human ß-globin chromatin domain.

Transcriptome analyses show a surprisingly large proportion of the mammalian genome is transcribed; much more than can be accounted for by genes and introns alone. Most of this transcription is non-coding in nature and arises from intergenic regions, often overlapping known protein-coding genes in sense or antisense orientation. The functional relevance of this widespread transcription is unknown. Here we characterize a promoter responsible for initiation of an intergenic transcript located approximately 3.3 kb and 10.7 kb upstream of the adult-specific human ß-globin genes. Mutational analyses in ß-YAC transgenic mice show that alteration of intergenic promoter activity results in ablation of H3K4 di- and tri-methylation and H3 hyperacetylation extending over a 30 kb region immediately downstream of the initiation site, containing the adult δ- and ß-globin genes. This results in dramatically decreased expression of the adult genes through position effect variegation in which the vast majority of definitive erythroid cells harbor inactive adult globin genes. In contrast, expression of the neighboring ε- and γ-globin genes is completely normal in embryonic erythroid cells, indicating a developmentally specific variegation of the adult domain. Our results demonstrate a role for intergenic non-coding RNA transcription in the propagation of histone modifications over chromatin domains and epigenetic control of ß-like globin gene transcription during development.

Advances in in vitro production of sheep embryos.

Sheep is an important livestock in the world providing meat, milk and wool for human beings. With increasing human population, the worldwide needs of production of sheep have elevated. To meet the needs, the assistant reproductive technology including ovine in vitro embryo production (ovine IVP) is urgently required to enhance the effective production of sheep in the world. To learn the status of ovine IVP, we collected some publications related to ovine IVP through PubMed and analyzed the progress in ovine IVP made in the last five years (2012-2017). We made comparisons of these data and found that the recent advances in ovine IVP has been made slowly comparable to that of ovine IVP two decades ago. Therefore, we suggested two strategies or approaches to tackle the main problems in ovine IVP and expect that the efficiency of ovine IVP could be improved significantly when the approaches would be implemented.

[Establishment of cell culture system of rough-legged buzzard and biological characteristic analysis on different tissue cells cultured in vitro].

In total, three different tissues from the rough-legged buzzard were obtained by culture and successfully cryopreserved and then recovered. During the subculture process, biological characteristics including as cell morphology, growth curve, cell adhesion rate, and karyotype were analyzed and compared, and overall all three kinds of tissue cells exhibited fibroblast-like growth. Oviduct-derived cells had the strongest adherent ability, followed by lung-derived cells and trachea-derived cells. The doubling times of lung-derived cells, trachea-derived cells, and oviduct-derived cells were 29.91±0.39 h, 33.18±0.21 h, and 30.67±0.28 h, respectively, with population doubling times 3.54±0.01, 4.52±0.02, and 4.38±0.03, respectively. Likewise, we noted the chromosome number of the rough-legged buzzard was 68, within the typical type of ZW. These results may potentially provide material and a basis for further research in the field, with the successful preservation of genetic information of rough-legged buzzard.

The Corfu deltabeta thalassemia deletion disrupts gamma-globin gene silencing and reveals post-transcriptional regulation of HbF expression.

The 7.2 kilobase (kb) Corfu deltabeta thalassemia mutation is the smallest known deletion encompassing a region upstream of the human delta gene that has been suggested to account for the vastly different phenotypes in hereditary persistence of fetal hemoglobin (HPFH) versus beta thalassemia. Fetal hemoglobin (HbF) expression in Corfu heterozygotes and homozygotes is paradoxically dissimilar, suggesting conflicting theories as to the function of the region on globin gene regulation. Here, we measure gamma- and beta-globin gene transcription, steady-state mRNA, and hemoglobin expression levels in primary erythroid cells cultured from several patients with Corfu deltabeta thalassemia. We show through RNA fluorescence in situ hybridization that the Corfu deletion results in high-level transcription of the fetal gamma genes in cis with a concomitant reduction in transcription of the downstream beta gene. Surprisingly, we find that elevated gamma gene transcription does not always result in a corresponding accumulation of gamma mRNA or fetal hemoglobin, indicating a post-transcriptional regulation of gamma gene expression. The data suggest that efficient gamma mRNA accumulation and HbF expression are blocked until beta mRNA levels fall below a critical threshold. These results explain the Corfu paradox and show that the deleted region harbors a critical element that functions in the developmentally regulated transcription of the beta-globin genes.