Role of chromosome stability and telomere length in the production of viable cell lines for somatic cell nuclear transfer

BACKGROUND: Somatic cell nuclear transfer (SCNT) provides an appealing alternative for the preservation of genetic material in non-domestic and endangered species. An important prerequisite for successful SCNT is the availability of good quality donor cells, as normal embryo development is dependent upon proper reprogramming of the donor genome so that embryonic genes can be appropriately expressed. The characteristics of donor cell lines and their ability to produce embryos by SCNT were evaluated by testing the effects of tissue sample collection (DART biopsy, PUNCH biopsy, post-mortem EAR sample) and culture initiation (explant, collagenase digestion) techniques. RESULTS: Differences in initial sample size based on sample collection technique had an effect on the amount of time necessary for achieving primary confluence and the number of population doublings (PDL) produced. Thus, DART and PUNCH biopsies resulted in cultures with decreased lifespans (<30 PDL) accompanied by senescence-like morphology and decreased normal chromosome content (<40% normal cells at 20 PDL) compared to the long-lived (>50 PDL) and chromosomally stable (>70% normal cells at 20 PDL) cultures produced by post-mortem EAR samples. Chromosome stability was influenced by sample collection technique and was dependent upon the culture's initial telomere length and its rate of shortening over cell passages. Following SCNT, short-lived cultures resulted in significantly lower blastocyst development (≤ 0.9%) compared to highly proliferative cultures (11.8%). Chromosome stability and sample collection technique were significant factors in determining blastocyst development outcome. CONCLUSION: These data demonstrate the influence of culture establishment techniques on cell culture characteristics, including the viability, longevity and normality of cells. The identification of a quantifiable marker associated with SCNT embryo developmental potential, chromosome stability, provides a means by which cell culture conditions can be monitored and improved

Exploiting Microfluidics for Extracellular Vesicle Isolation and Characterization: Potential Use for Standardized Embryo Quality Assessment

Recent decades have seen a growing interest in the study of extracellular vesicles (EVs), driven by their role in cellular communication, and potential as biomarkers of health and disease. Although it is known that embryos secrete EVs, studies on the importance of embryonic EVs are still very limited. This limitation is due mainly to small sample volumes, with low EV concentrations available for analysis, and to laborious, costly and time-consuming procedures for isolating and evaluating EVs. In this respect, microfluidics technologies represent a promising avenue for optimizing the isolation and characterization of embryonic EVs. Despite significant improvements in microfluidics for EV isolation and characterization, the use of EVs as markers of embryo quality has been held back by two key challenges: (1) the lack of specific biomarkers of embryo quality, and (2) the limited number of studies evaluating the content of embryonic EVs across embryos with varying developmental competence. Our core aim in this review is to identify the critical challenges of EV isolation and to provide seeds for future studies to implement the profiling of embryonic EVs as a diagnostic test for embryo selection. We first summarize the conventional methods for isolating EVs and contrast these with the most promising microfluidics methods. We then discuss current knowledge of embryonic EVs and their potential role as biomarkers of embryo quality. Finally, we identify key ways in which microfluidics technologies could allow researchers to overcome the challenges of embryonic EV isolation and be used as a fast, user-friendly tool for non-invasive embryo selection

The oxidative stress adaptor p66Shc is required for permanent embryo arrest in vitro

<p>Abstract</p> <p>Background</p> <p>Excessive developmental failure occurs during the first week of <it>in vitro </it>embryo development due to elevated levels of cell death and arrest. We hypothesize that permanently arrested embryos enter a stress-induced "senescence-like" state that is dependent on the oxidative stress-adaptor and lifespan determinant protein p66Shc. The aim of this study was to selectively diminish p66Shc gene expression in bovine oocytes and embryos using post-transcriptional gene silencing by RNA-mediated interference to study the effects of p66Shc knockdown on <it>in vitro </it>fertilized bovine embryos.</p> <p>Results</p> <p>Approximately 12,000–24,000 short hairpin (sh)RNAi molecules specific for p66Shc were microinjected into bovine germinal vesicle stage oocytes or zygotes. Experiments were comprised of a control group undergoing IVF alone and two groups microinjected with and without p66Shc shRNAi molecules prior to IVF. The amount of p66Shc mRNA quantified by Real Time PCR was significantly (P < 0.001) lowered upon p66Shc shRNAi microinjection. This reduction was selective for p66Shc mRNA, as both histone H2a and p53 mRNA levels were not altered. The relative signal strength of p66Shc immuno-fluorescence revealed a significant reduction in the number of pixels for p66Shc shRNAi microinjected groups compared to controls (P < 0.05). A significant decrease (P < 0.001) in the incidence of arrested embryos upon p66Shc shRNAi microinjection was detected compared to IVF and microinjected controls along with significant reductions (P < 0.001) in both cleavage divisions and blastocyst development. No significant differences in p66Shc mRNA levels (P = 0.314) were observed among the three groups at the blastocyst stage.</p> <p>Conclusion</p> <p>These results show that p66Shc is involved in the regulation of embryo development specifically in mediating early cleavage arrest and facilitating development to the blastocyst stage for in vitro produced bovine embryos.</p

Skin Lipoma in an Arabian Leopard (Panthera paradus nimr) Acta Scientiae Veterinariae

ABSTRACT Background: The Arabian leopard (Panthera paradus nimr) is the largest living felid in the arid Arabian Peninsula and classifi ed on the IUCN red list as critically endangered. Unlike felids, neoplasia prevalence in canids such as benign lipoma and malignant liposarcoma has been long and well documented. Only until recently a plethora of reports emerged demonstrating that neoplasia occurrence in wild exotic felids is prevailed more than expected. Soft tissue tumors arise from fatty cells form either a benign lipoma or a dangerously malignant liposarcoma. Alarming though, such cellular transformation might endanger the life of an already endangered animal. Case: An intact Arabian male leopard living in captivity at the Oman wildlife animal breeding center (N23.70 E58.09 A5.80 m) aged approximately 18 years and weight 31 kg was admitted to the veterinary clinic for semen collection and routine physical examination. The animal was identifi ed with two large adjacent subcutaneous masses on the upper rear left limb, clinically resembling that of a lipomatosis. Only one large tissue mass was surgically excised from the base with no incident of bleeding. Gross examination revealed a soft, smooth, rubbery, homogeneous, lack of internal fl uid and whitish color lobule. Morphometry measurement of the mass shows that the weight, diameter, circumference, thickness and surface area were 3.6 gm, 2.6 cm, 10.2 cm, 3 cm and 17.8 cm 3 respectively. On visual examination, neither mucin fl uid nor mucosal ulcerations were detected. Microscopically, dark discrete spots were observed on the anterior central and periphery of the mass surface outgrowth. Moreover, histopathological diagnoses with haematoxylin and eosin (HE), masson fontana (MF) and elastic verrhof van giesson (EVG) revealed normal nuclear and non-granular cytoplasm resembling that of a fatty cell originating from a fat adipose tissue. Adipocytes had reasonable amounts of cytoplasm and well defi ned borders. The nuclei were round to oval shape and no cells were found to be multinucleated. No evidence of high nuclear cytoplasmic ratio was observed. Few lymphocytes and plasma cells were present with no visible lymphatic vessels. Taken together, the lesion was diagnosed as a lobulated soft mass resembling that of an adipose tissue, specifi cally a benign neoplastic lipoma. Discussion: To date not a single report describes maladies in big cats from arid regions. This is the fi rst study to demonstrate the occurrence of neoplasia in a wild felid namely; the Arabian leopard. Additionally, while recent reports have shown neoplasia occurrence in the Panthera subspecies in tropical, polar and temperate zones, this is the fi rst report to manifest the disease in an arid region. The increase in neoplasm frequency in exotic felids is a concerning fact as numerous members of the Panthera family including the Arabian leopard are classifi ed by the IUCN as endangered or critically endangered species. With less than 200 animals in the wild, only 14 founder individuals in captivity and an aged female population the occurrence of lipoma tumors in the Arabian leopard is a worrisome sign. Taken together, the data suggests the rise of uncommon diseases in carnivores and ubiquitously around different climate zones of the world. Thus highlights the importance of routine physical examinations, investing substantially in diagnostic equipment and healthcare endowment in captive exotic felids

Recent acquisition of imprinting at the rodent Sfmbt2 locus correlates with insertion of a large block of miRNAs.

BACKGROUND: The proximal region of murine Chr 2 has long been known to harbour one or more imprinted genes from classic genetic studies involving reciprocal translocations. No imprinted gene had been identified from this region until our study demonstrated that the PcG gene Sfmbt2 is expressed from the paternally inherited allele in early embryos and extraembryonic tissues. Imprinted genes generally reside in clusters near elements termed Imprinting Control Regions (ICRs), suggesting that Sfmbt2 might represent an anchor for a new imprinted domain. RESULTS: We analyzed allelic expression of approximately 20 genes within a 3.9 Mb domain and found that Sfmbt2 and an overlapping non-coding antisense transcript are the only imprinted genes in this region. These transcripts represent a very narrow imprinted gene locus. We also demonstrate that rat Sfmbt2 is imprinted in extraembryonic tissues. An interesting feature of both mouse and rat Sfmbt2 genes is the presence of a large block of miRNAs in intron 10. Other mammals, including the bovine, lack this block of miRNAs. Consistent with this association, we show that human and bovine Sfmbt2 are biallelic. Other evidence indicates that pig Sfmbt2 is also not imprinted. Further strengthening the argument for recent evolution of Sfmbt2 is our demonstration that a more distant muroid rodent, Peromyscus also lacks imprinting and the block of miRNAs. CONCLUSIONS: These observations are consistent with the hypothesis that the block of miRNAs are driving imprinting at this locus. Our results are discussed in the context of ncRNAs at other imprinted loci. Accession numbers for Peromyscus cDNA and intron 10 genomic DNA are [Genbank:HQ416417 and Genbank:HQ416418], respectively.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Identification of familial networks reveals sex-specific density dependence in the dispersal and reproductive success of an endangered ungulate

Density is an important demographic parameter that is commonly overlooked in studies of wild populations. Here, we examined the effects of variable spatially explicit density on a range of demographic parameters in a wild population of a cryptic ungulate, boreal woodland caribou (Rangifer tarandus caribou). Using non-invasive genetic sampling, we applied spatial capture–recapture methods with landscape covariates to estimate the density of boreal woodland caribou across a 108,806 km2 study area. We then created a familial network from the reconstructed parent–offspring relationships to determine whether spatial density influenced sex-specific individual reproductive success, female pregnancy status, and dispersal distance. We found that animal density varied greatly in response to land cover types and disturbance; animal density was most influenced by landscape composition and distance to roads varying from 0 in areas with &gt;20% deciduous cover to 270 caribou per 1,000 km2 in areas presenting contiguous older coniferous cover. We found that both male and female reproductive success varied with density, with males showing a higher probability of having offspring in higher-density areas, and the opposite for females. No differences were found in female pregnancy rates occurring in high- and low-density areas. Dispersal distances varied with density, with offspring moving shorter distances when parents were found in higher-density areas. Familial networks showed lower-closeness centrality and lower-degree centrality for females in higher-density areas, indicating that females found in higher-density areas tend to be less broadly associated with animals across the range. Although high-density areas do reflect good-quality caribou habitat, the observed decreased closeness and degree centrality measures, dispersal rates, and lower female recruitment rates suggest that remnant habitat patches across the landscape may create population sinks

Tissue sampling methods and standards for vertebrate genomics

The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences across major vertebrate groups. Standardized procedures in acquiring high quality DNA and RNA and establishing cell lines from target species will facilitate these initiatives. We provide a legal and methodological guide according to four standards of acquiring and storing tissue for the Genome 10K Project and similar initiatives as follows: four-star (banked tissue/cell cultures, RNA from multiple types of tissue for transcriptomes, and sufficient flash-frozen tissue for 1 mg of DNA, all from a single individual); three-star (RNA as above and frozen tissue for 1 mg of DNA); two-star (frozen tissue for at least 700 μg of DNA); and one-star (ethanol-preserved tissue for 700 μg of DNA or less of mixed quality). At a minimum, all tissues collected for the Genome 10K and other genomic projects should consider each species’ natural history and follow institutional and legal requirements. Associated documentation should detail as much information as possible about provenance to ensure representative sampling and subsequent sequencing. Hopefully, the procedures outlined here will not only encourage success in the Genome 10K Project but also inspire the adaptation of standards by other genomic projects, including those involving other biota

Tissue Sampling Methods and Standards for Vertebrate Genomics

The recent rise in speed and efficiency of new sequencing technologies have facilitated high-throughput sequencing, assembly and analyses of genomes, advancing ongoing efforts to analyze genetic sequences across major vertebrate groups. Standardized procedures in acquiring high quality DNA and RNA and establishing cell lines from target species will facilitate these initiatives. We provide a legal and methodological guide according to four standards of acquiring and storing tissue for the Genome 10K Project and similar initiatives as follows: four-star (banked tissue/cell cultures, RNA from multiple types of tissue for transcriptomes, and sufficient flash-frozen tissue for 1 mg of DNA, all from a single individual);three-star (RNA as above and frozen tissue for 1 mg of DNA); two-star (frozen tissue for at least 700 μg of DNA); and one-star (ethanol-preserved tissue for 700 μg of DNA or less of mixed quality). At a minimum, all tissues collected for the Genome 10K and other genomic projects should consider each species’ natural history and follow institutional and legal requirements. Associated documentation should detail as much information as possible about provenance to ensure representative sampling and subsequent sequencing. Hopefully, the procedures outlined here will not only encourage success in the Genome 10K Project but also inspire the adaptation of standards by other genomic projects, including those involving other biota

Proceedings of the IETS 2009 Post-Conference Symposium: implementation of artificial insemination in companion animals, non-domestic and endangered species (CANDES)

While assisted reproductive technologies (ART) have had an enormous impact on the breeding and understanding of reproduction in domestic and laboratory species as well as humans, progress is poor in many companion animals, non-domestic and endangered species (CANDES). The first draft program for this meeting was composed in April 2006 when members of the CANDES Parent Committee of the IETS (www.omahazoo.com/IETS/CANDEShomepage.htm) coined the idea of holding workshops to inform IETS members of realistic expectations when extrapolating ART from livestock and humans to CANDES. As co-chairs of the Research and Technology subcommittees, we and our colleagues, Rebecca Krisher and Monique Paris, are proud to deliver the first of these workshops entitled "Implementation of Artificial Insemination in CANDES"