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The gene-editing (GE) is of great potential to be applied to agriculture and biomedicine. The organ waiting patients may be treated with tailored made xenografts derived from porcine blastocyst that the original key developing gene has been knockout by GE but complemented with patient's induced pluripotent stem cells (iPSC). This study used day 6 porcine morulae (M) or blastocysts (BC) from chemical activated parthenogenesis and each embryo was microinjected with 10 to 15 Formosan monkey or porcine GFP-iPSCs (GFP-FMiPSC or GFP-piPSC) to generate the inter- or intra-species chimera. The embryos were then cultured in PZM-5 at 38.5°C/5% CO2 for 24 and 48 h to assess their development. Results showed that the survival rates of GFP-FMiPSC/BC (n = 61) and GFP-piPSC/BC (n = 60) were similar at 24 h (73.4 v.s 73.3%, respectively) yet significantly different at 48 h (69.2 ± 11.2 v.s. 53.3 ± 8.9%, respectively) (P < 0.01). On day 6, M might be over aging developed and appeared less viability and fewer developed rate (7.3 and 7.4%) to BC. [ABSTRACT FROM AUTHOR]

Frozen semen is traditionally used for pig genetic resource preservation; however, embryonic vitrification (VF) is also a well-established and available method. We have established porcine blastocyst (BC) VF, and the purpose of this study was to compare the survival rates (SR) of parthenogenetic BC (61 embryos/6 lots) and morula (M; 101 embryos/7 lots) from different chemically activated (day 6 ~ 7). The embryos were treated with basal medium [BM, M199 + 20% FBS + 0.5 M sucrose (S) ] and fallowed by equilibrating [BM + 7.5% ethylene glycol (EG) + 7.5% propylene glycol (PG) ] and VF (BM + 15% EG + 15% PG) for 2 min and 30 sec, respectively. After 1-month frozen, the embryos were thawed by 3 steps which with M199 containing 20 % FBS, and added with 1.0 M, 0.5 M, and 0.0 M S for 1, 3, and 5 min, respectively. After thawed, all embryos were cultured in PZM-5 in 5% CO2/ 38.5 V incubator for 24 h and the SR were assessed. Results showed that the SR of M vs BC were 59.3 ± 5.6% vs. 78.3 ± 5.5%, respectively, with significant differences (P < 0.05). Additionally, only 16.1 ± 6.6% M of those survival were developed to BC stage. These results suggested that blastocyst is better than morula by VF for porcine genetic resources cryopreservation. [ABSTRACT FROM AUTHOR]

The electroanatomic mapping systems (EMS) have been commonly used in difficult for curing arrhythmia and myocardial infarction. However, more practical exercises are required to increase the success rate. The anatomy of miniature pig is similar to human and recognized as a ideal model to practice the EMS and interventional ablation. Lee Sung pig, a miniature pig was developed in National Taiwan University and never been used as a model to examine the EMS. The aim of this study was to evaluate the EMS by using the basket catheter in Lee Sung pig model. The result showed the EMS was successfully used on adult Lee Sung pig. In addition, the calculated electrogram showed success of radiofrequency ablation of ventricle by EMS. These results show Lee Sung pig is an idea model for practical exercise the EMS. [ABSTRACT FROM AUTHOR]

Miniature pig (Sus Scrofa) becomes as an important ocular animal model for the ophthalmology research due to the anatomic and physiologic similarities to human. Lee Sung pig is a miniature pig breed originated in Taiwan. However, absent of ocular structure leads difficult be appiled in occular research. In present study, Magnetic Resonance Imaging (MRI) was applied for the ocular structure assessment of different developmental stages of Lee Sung pig. The detailed description of the structure and size of the different developmental stage oculars structure will be of great help to future ophthalmology application of Lee Sung pig. [ABSTRACT FROM AUTHOR]

In response to the liberalization of world trade, develop unique livestock product and expand niche markets become a key to success. Taiwan native black pig included Lanyu pig, Taoyuan pig and native black pig reared in the Pingtung area. We used 19 microsatellite markers to do an assignment test of the native black pig with exotic pig breeds, we called the native black pig which has a distinctive characteristic 'LiuDui' native black pig and it has a good evaluation in the market. In order to provide a DNA marker for 'LiuDui' native black pig, 10 markers with high PIC value were chosen to do an assignment test to detect the individual identification ability by ranking their F-statistics FST), Heterozygosity (He), Allelic number (K), and relative discriminatory power (D-score). Higher effective and accuracy marker will be picked out and also showed the order of using the marker. The result of this study can be used as genetic monitoring and product traceability for native black pig. [ABSTRACT FROM AUTHOR]

Goat β-casein (CSN2) promoter has been extensively used to derive expression of recombinant therapeutic protein in transgenic goats; however, little direct evidence exists for signaling molecules and the cis-elements of goat CSN2 promoter in response to lactogenic hormone stimulation in goat mammary epithelial cells. Here, we use an immortalized caprine mammary epithelial cell line (CMC) to search for evidence of the above. Serial 5'-flanking regions deleted of promoter and intron 1 in goat CSN2 (-4,047 to +2,054) driven by firefly luciferase reporter gene were constructed and applied to measure promoter activity in CMC. The intron 1 region (+393 to +501) significantly decreased basal activity of the promoter. This finding contradicts other studies of the role of intron 1. The signal transducer and activator of transcription (STAT)5a played a significant role in activating promoter activity by prolactin stimulation. Hydrocortisone enhanced and prolonged the activity of STAT5a and promoter in CMC, but was independent of the glucocorticoid receptor response element. The minimum length of the CSN2 promoter segment in response to lactogenic stimulation was confirmed by 5' serial deletions. A cis-element located from -300 to -90 in proximal goat CSN2 promoter that is absent in bovine and human CSN2 promoter was newly identified. We demonstrated the presence of a STAT5a binding site (-102 to -82) and preservation of the guanosine nucleotide at position -90 based on responses to the presence of lactogenic hormone using internal deletions and point mutations of the predicted STAT5a binding site, and chromatin immunoprecipitation assay. Together, these findings demonstrate that the proximal -300 bp of goat CSN2 promoter containing the STAT5a binding site (-102 to -82) is the response element for lactogenic hormone stimulation. Additionally, intron 1 may be required for tissue or developmental stage-specific expression in mammary gland. The role of the far-distal regions of goat CSN2 promoter in high-level lactogenic hormone induction and specific expression require further examination. [ABSTRACT FROM AUTHOR]

The aim of this article is to demonstrate and characterize caprine mammary epithelial cells (CMC) immortalized with human telomerase reverse transcriptase (hTERT) gene. Five immortalized CMCs were assigned to either myoepithelial or luminal epithelial groups based on their morphology and expression of cell lineage-specific intermediate filaments. Telomeric repeat amplification protocol revealed various telomerase activities in CMCs associated with their distinct proliferation potential. Karyotypic analysis showed three CMCs retained their modal Capra hircus chromosome number (2n = 60), whereas the remaining two CMCs were abnormal at 2n = 19 and 2n = 36. CMCs with abnormal karyotypes lost p53 protein after chemical-induced DNA damage and showed anchorage-independent growth in soft agar assay. In terms of functional differentiation, luminal CMCs organized into alveolus-like structures when grown in Matrigel. Furthermore, αs1- and β-casein gene was induced in luminal CMCs in response to lacto-hormones stimulation. Together these results showed that hTERT-immortalized CMCs retained major characteristics of mammary epithelial cells, and stability of the genome is required for maintaining normal mammary epithelium function. Application of CMCs can provide valuable models to study alveologenesis and lactogenesis of mammary epithelium and test the feasibility of recombinant constructs designed for the generation of transgenic livestock., (© 2011 Blackwell Verlag GmbH.)

Gas7, a growth arrest-specific gene first isolated from serum-starved NIH3T3 cells, is expressed abundantly in the brain and is essential for the outgrowth of neurites from cultured cerebellar neurons. Here, we report the existence of a Gas7-related cDNA, designated Gas7-cb, isolated from the mouse cerebellum, and we report the finding that Gas7-cb transcripts and protein are expressed at different locations than those of Gas7. Gas7-cb cDNA differs from the Gas7 cDNA only in the 5' region. Its encoded protein shares the same 320 amino acids in its C-terminus with those of Gas7. Analyses of the RNA and protein expression of Gas7-cb and Gas7 by RNase protection assay and Western blot indicated that while Gas7 expression is predominant in the cerebrum and in growth-arrested NIH3T3 fibroblasts, Gas7-cb expression is predominant in the cerebellum. Characterization of Gas7 and Gas7-cb RNAs and of the genomic structure of murine Gas7 cloned in a bacterial artificial chromosome indicated that the Gas7 gene spans more than 60 kb and consists of at least 15 exons. The 5'-terminus of Gas7-cb is located at exon 6a, which is absent in Gas7 transcripts but is retained in its entirety in Gas7-cb transcripts, resulting in the presence of a unique 20-amino-acid sequence at the N-terminus of the Gas7-cb protein. Our results show that the Gas7 gene encodes two Gas7 isoforms, Gas7 and Gas7-cb, whose expression is differentially regulated within mouse brain., (Copyright 1999 Academic Press.)

Growth arrest-specific (gas) genes are expressed preferentially in cells that enter a quiescent state. gas7, which we identified in serum-starved murine fibroblasts, is reported here to be expressed in vivo selectively in neuronal cells of the mature cerebral cortex, hippocampus, and cerebellum. gas7 transcripts encode a 48-kDa protein containing a structural domain that resembles sequences of OCT2, a POU transcription factor implicated in neuronal development, and synapsins, which have a role in modulating neurotransmitter release. Using in situ hybridization and immunocytochemical analysis, we show that GAS7 expression occurs prominently in cerebellar Purkinje cells and that inhibition of production in terminally differentiating cultures of embryonic murine cerebellum impedes neurite outgrowth from maturing Purkinje cells. Conversely, GAS7 overexpression in undifferentiated neuroblastoma cell cultures dramatically promotes neurite-like outgrowth. Collectively, our results provide evidence for an association between expression of this gas gene and neuronal development.