Fertile offspring derived from mouse spermatogonial stem cells cryopreserved for more than 14 years
ABSTRACT: To evaluate the possible deleterious affects prolonged cryopreservation and assisted reproductive technologies may have on the DNA of F0 progeny, we used array-based comparative genomic hybridization (CGH) for analysis of genomic DNA. ICSI-derived progeny were generated from ♂ (ZFLacZ sperm) x ♀ (DBA x C57BL/6 oocyte), where the ZFLazZ transgenic mice were originally generated from an SJL x C57BL/6 background.
Project description:Seventy blastomeres from fourteen frozen-thawed supernumerary human preimplantation embryos were disassociated and genomic was amplified using Multiple Displacement Amplification. BAC array-CGH was performed on the amplified products. BAC array-CGH on single blastomeres amplified by Multiple Displacement Amplification.
Project description:The oncomir microRNA-125b (miR-125b) is up-regulated in a variety of human neoplastic blood disorders and constitutive up-regulation of miR-125b in mice can promote myeloid and B cell leukemia. We found that miR-125b promotes myeloid and B cell neoplasm by inducing tumorigenesis in hematopoietic progenitor cells. Our study demonstrates that miR-125b induces myeloid leukemia by enhancing myeloid progenitor output from stem cells as well as inducing immortality, self-renewal, and tumorigenesis in myeloid progenitors. Through functional and genetic analyses, we demonstrated that miR-125b induces myeloid and B cell leukemia by inhibiting IRF4 but through distinct mechanisms; it induces myeloid leukemia through repressing IRF4 at the mRNA level without altering the genomic DNA and induces B cell leukemia via genetic deletion of the gene encoding IRF4. The cancer myeloid (Cd11b+ sorted) and B cells (CD19+ sorted) were harvested from mice that over-express miR-125b. The genomic DNA was extracted from these cells. A total of 4 cancer samples (Two myeloid cancer samples and two B cell cancer samples) were analyzed. As control, genomic DNA from cells harvested from healthy C57bl/6 mice were harvested.
Project description:Expression profiling of sheep born to Australian industry sires with high and low genetic merit (Estimated Breeding Values or EBVs) for eye muscle depth (EMD). Progeny (40) from six Poll Dorset sires representing well defined extremes of EBVs for Eye Muscle Depth (low EBV EMD and high EBV EMD) were selected for analysis. The six sires were Australian industry sires with three sires representative of low EBV EMD and three representing high EBV EMD. Microarrays were used for transcription profiling of skeletal muscle samples taken from 40 individual progeny belonging to six Poll Dorset industry sires, with 3 sires extreme for high and 3 sires extreme for low muscling (based on EBV for EMD). Sheep longissimus dorsi (LD) skeletal muscle samples were collected from 40 individual progeny belonging to six Poll Dorset industry sires, with 3 sires extreme for high and 3 sires extreme for low muscling (based on EBV for EMD).
Project description:Many long-lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high-throughput RNA sequencing (RNA-Seq) to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently-defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥ 5-fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell-associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes composed the ancestral circuitry underlying the control of stem cell self-renewal and pluripotency. Three planarian tissues were analyzed: stem cells, stem cell progeny, and differentiated tissues. For the manuscript: Labbe, RM, et. al., 2012, Stem Cells
Project description:Effects of heat priming applied to the first generation on tolerance of the successive generation to post-anthesis high temperature stress were investigated. Compared with the progeny of non-heat primed plants (NH), the progeny of heat-primed plants (PH) presented higher grain yield, leaf photosynthesis and activities of antioxidant enzymes and lower cell membrane damage under high temperature stress. In the transcriptome profile, 1430 probes showed obvious difference in expression between PH and NH. These genes were those of signal transduction, transcription, energy, defense, and protein destination and storage, respectively. Pot experiments (25 cm in diameter and 22 cm in depth) were conducted at the Experimental Station of Nanjing Agricultural University, Nanjing, China (32˚30ʹN and 118˚42ʹE). During the first generation, wheat plants were divided into two groups: one group were not primed (N) while the other were heat-primed (P) at pre-anthesis (at a day/night temperature of 32/28˚C for two days at the seven-leaf stage and the nine-leaf stage) and post-anthesis (34/30˚C for seven days at the 10th day after anthesis). At maturity, seeds from both groups were harvested. Seedlings (the progeny generation) from each group seed were further divided two sub-groups: one was subjected to high temperature stress at a day/night temperature of 34/30˚C, while the other was set at 26/22˚C for six days from the 10th day after anthesis (DAA). During both priming and high-temperature stress, plants under different treatments were moved into sepatarate growth chambers at preset temperatures. Thereafter, four treatments were established: NC, progeny of non-primed plants without post-anthesis high temperature stress; NH, progeny of non-primed plants with post-anthesis high temperature stress; PC, progeny of primed plants without post-anthesis high temperature stress; and PH, progeny of primed plants with post-anthesis high temperature stress.
Project description:Competent oocytes can be discriminated by BCB staining. Positive stained oocytes are considered more competent than BCB negative oocyte, and injection of BCB+ oocyte extracted mitochondria into BCB negative oocytse can increase fertilisation and blastocyst rate. Here we have analysed the impact of mitochondrial supplementation on subsequent blastocyst transcriptome using agilent one color microarray that is specificly design to study the porcine embryo preimplantation period. Blastocysts were produced by intra cytoplasmic sperm injection (ICSI) from BCB positive and BCB negative oocytes as well as BCB negative oocytes supplemented with mitochondrial extract during ICSI (mICSI), and 3-4 single blatocyst transcriptomes were analysed for each group. 3-4 single blastocysts were analysed at the RNA level after whole transcriptome amplification, and level of gene expression was compared between groups, i.e ICSI BCB+ blastocysts (4), ICSI BCB- blastocysts (3) and mICSI BCB- blastocysts (4).
Project description:Human cord blood CD34+ cells transduced with control of IK6 lentivirus were transplanted into NSG mice. Control and IK6 transduced CD34+38- progeny were acquired 10 weeks later by FACS Comparison of control and IK6+ human CD34+38- progenitor cells
Project description:The goal of this study was to analyze global gene expression in specific populations of nociceptor sensory neurons, the neurons that detect damaging/noxious stimuli. The dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia are anatomically distinct peripheral sensory ganglia that contain nociceptors which innervate skin, gut, lungs, and other distinct organ tissues. We used flow cytometry to purify nociceptors from these ganglia and profiled their global gene expression signatures to compare gene expression between these different anatomically distinct nociceptors. Nav1.8-Cre were bred with Rosa26-TdTomato to generate Nav1.8-Cre/R26-TdTomato reporter progeny, where all peripheral nociceptor neurons are genetically marked with red fluroescence due to specific expression of the TTX- resistant sodium channel Nav1.8. Lumbar region dorsal root ganglia (DRG), trigeminal ganglia, and nodose ganglia were dissected from mice (3 mice were pooled/sample). Highly red fluorescent neurons were Facs purified, RNA extracted, and processed for microarray analysis.
Project description:Originating from Northeast Asia, the Pacific oyster Crassostrea gigas has been introduced into a large number of countries for aquaculture purpose. Following introduction, the Pacific oyster has turned into an invasive species in an increasing number of coastal areas, notably in Northern Europe. To explore adaptation on reproductive traits of population considered as invasive, we set up a common garden experiment based on the comparison of progenies from two populations of Pacific oyster sampled in France and Denmark. A female-biased sex-ratio and a higher condition index were observed in the Danish progeny, possibly reflecting an evolutionary reproductive strategy to increase the potential success of natural recruitment in recently settled population. Using multifarious statistical approaches and accounting for sex differences we identified several genes differentially expressed between the Danish and French progenies, and with an intermediate expression level in hybrids (additive behavior). Candidate transcripts included mRNA coding for sperm quality and insulin metabolism known to be implicated in coordinated control of reproduction. Our results suggest adaptation of invasive populations during expansion acting on reproductive traits, and in particular on a female-biased sex-ratio, fertility and gamete quality. A common garden experiment was performed in order to compare progenies from two populations of Pacific oyster sampled in France and Denmark and their hybrids. Progenies were reared under standard hatchery and nursery conditions until gonadal maturation. The employed arrays were Agilent 60-mer 4x44K custom microarrays, containing 31,918 C. gigas ESTs, designed by Dheilly et al. (2011).