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Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with memory loss and cognitive impairments. An AD transgenic (Tg) pig model would be useful for preclinical testing of therapeutic agents. In this study, we report the use of somatic cell nuclear transfer (SCNT) to produce transgenic pigs over-expressing the human AD related genes (APP, APPswedish, Presenilin 1, and Tau). Transgenic embryos were generated by SCNT of from ear fibroblasts expressing AD genes. A total of 1808 (average 258) SCNT embryos were transferred into 7 recipients. Pregnancy was successfully maintained in one recipient. We obtained 1 cloned male piglet from a surrogate gilts and the weight of piglet was 935 g. But, the male piglet died two days after birth. The piglet expressed AD related genes by PCR and western blot analysis. Transgenes were expressed in multiple tissues, and at especially high levels in brain. AD Tg pig might be very useful for studying the disease and for testing new therapeutics in preclinical studies of human AD.
This study was performed to determine the developmental potentials of nuclear transfer (NT) embryos using life-span extended cells transfected with a foreign gene as donor cells. A life-span extended bovine embryonic fibroblast cell line was transfected with an expression vector in which the human type II collagen (BOMAR) and ear fibroblasts were used as a donor cell. Cytogenetic analysis was performed to analyze the chromosomal abnormality of donor cells. The fusion rate of 1.8 kV/cm for 15 ??sec given twice was significantly higher than that of other groups (p<0.05) and the embryos lysed were significantly higher after 1.8 kV/cm for 20 ??sec given once compared to other groups (p<0.01). The blastocyst development in the ear cell group was statistically significant compared to both BOMAR groups (p<0.01). Both BOMAR groups cultured more than 40 passages (>40 passages) had a lower number of chromosomes; however, fresh granulosa cell (GC) and BOMAR groups cultured less than 20 passages had normal chromosome numbers. Both >40 passages BOMAR groups had numerous obscure debris in metaphase spreads. The transfected foreign gene was expressed in all BOMAR groups, but not in the GC group. Based on these results, the lower developmental potential of NT embryos using life-span extended donor cells transfected with a foreign gene might be a cause of chromosomal abnormality in donor cells.
Understanding the sophisticated imprinting pattern is the first challenge for deciphering the molecular mechanism of an imprinted gene. However, the determination of imprinted genes, and their methylation status in pigs, remains inefficient at present. Herein, for the first time, we have made an attempt to illustrate the complex imprinting pattern of porcine GNAS complex locus by an efficient strategy using RNA-seq analysis of parthenogenetic fetuses containing no paternal allele, but two maternal alleles. RNA-seq results showed that Nespas, Gnasxl, and Exon 1A genes expressed at extremely lower levels (p<0.05) in parthenogenetic fetuses compared to the normally fertilized fetuses, suggest these three genes are paternally-expressed. Whereas, the expression levels of Gnas were similar in both groups, suggesting biallelic expression of Gnas. The parental-specific expression of these genes was further confirmed by quantitative real-time PCR. Bisulfite sequencing revealed that the maternally derived Nespas promoter was methylated, but the paternal chromosome was not methylated. Our finding provided full-scale imprinting information on porcine GNAS locus for the first time. Furthermore, our study is useful for identify novel imprinted genes, confirm the previously known imprinted genes, and determine imprinting pattern efficiently.
Hwang, Kyu-Chan,Cho, Seong-Keun,Lee, Seong-Hoon,Park, Jong-Yi,Kwon, Deug-Nam,Choi, Yun-Jung,Park, Chankyu,Kim, Jae-Hwan,Park, Keun-Kyu,Hwang, Seongsoo,Park, Soo-Bong,Kim, Jin-Hoi Wiley-Liss, Inc. 2009 Developmental dynamics Vol.238 No.7
<P>Previously, we have successfully produced nine cloned piglets using Duroc donor cells. Among these clones, one showed distinct depigmentation of the skin and hair color during puberty. In this study, we selected a clone with depigmentation to investigate the etiology of the anomaly in somatic cell nuclear transfer. We hypothesized that genes related to Waardenburg syndrome (Mitf, Pax-3, Sox-10, Slug, and Kit) are closely associated with the depigmentation of pig, which was derived from somatic cell nuclear transfer (scNT). Total RNA was extracted from the ear tissue of affected and unaffected scNT-derived pigs, and the transcripts encoding Mitf, Pax-3, Sox-10, and Slug, together with the Kit gene, were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed. The cDNA sequences from the scNT pig that showed progressive depigmentation did not reveal a mutation in these genes. Although we did not find any mutations in these genes, expression of the genes implicated in Waardenburg syndrome was severely down-regulated in the affected scNT pig when compared with unaffected scNT pigs. This down-regulation of gene expression may result in a previously undescribed phenotype that shows melanocyte instability, leading to progressive loss of pigmentation. Developmental Dynamics 238:1701–1708, 2009. © 2009 Wiley-Liss, Inc.</P>
Cyclin B1 is known to reflect the M-phase promoting factor (MPF), a universal regulator of G2/M-phase transition, activity during the process of oocytes maturation. To investigate whether culture condition affects the maturation rate and the expression of cyclin B1 protein, bovine immature oocytes are stimulated and cultured according to the following protocols: Experiment 1: denuded oocytes (denude) only, COC only, denuded oocytes + granulosa cells (denude + GCs) and COC + GCs; Experiment 2: no-activation (control), 7% ethanol for 5 min and 10 l/ml ionomycin for 5 min at immediately before maturation. The maturation rates of denude and no-activation group were significantly lower in both experiments (P<0.05), respectively. Co-culture or stimulation method in bovine immature oocytes culture increases the cyclin B1 expression significantly in both experiments (P<0.05). Based on these results, culture condition affects the maturation rate and the expression of cyclin B1 protein during the first meiotic maturation in bovine immature oocytes.