Analysis of proteomic profiling of mouse embryonic stem cells derived from fertilized, parthenogenetic and androgenetic blastocysts

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation embryos. ESCs exhibit true pluripotency, i.e., the ability to differentiate into cells of all three germ layers in the developing embryo. We used 2-DE MALDI-TOF/TOF to identify differentially expressed proteins among three types of mouse embryonic stem cells (ESCs) derived from ferti-lized, parthenogenetic, and androgenetic (fESC, pESC and aESC, respectively) blastocysts. We detected more than 800 proteins on silver- stained gels of whole protein extracts from each type of ESC. Of these, 52 differentially expressed proteins were identified by the MALDI–TOF/TOF analyzer, including 32 (fESCs vs. pESCs), 28 (fESCs vs. aESCs) and 17 (pESCs vs. aESCs) prominent proteins with significantly higher or lower expression relative to the comparison cells. Among the 32 proteins from fESCs, 12 were significantly increased in expression and 20 were reduced in expression in fESCs com-pared with pESCs. Similarly, 10 of 28 and 8 of 17 proteins were more highly expressed in fESCs and pESCs compared with aESCs, respectively. In contrast, 18 of 28 and 9 of 17 proteins were reduced in expression in fESCs and pESCs compared with aESCs, respectively. Of the eight protein candidates in fESCs, four were in-creased and four were decreased in expression relative to both pESCs and aESCs in the 2-DE analysis. Differential expression of these pro-teins were confirmed by mRNA expression analysis using real time RT-PCR. For three pro-teins, ANXA5, CLIC1 and SRM, Western blot analysis corroborated the expression patterns indicated by the 2-DE results. ANXA5 and CLIC1 were increased in expression and SRM was de-creased in expression in fESCs compared with both pESCs and aESCs. The differentially ex-pressed proteins identified in the present study warrant further investigation towards the goal of their potential application in ESC therapy.

Generation of pigs with humanized type Ⅱ collagen by precise human COL2A1 gene knock-in

Osteoarthritis(OA)is the most common chronic disease,characterized by progressive cartilage breakdown,subchondral bone sclerosis,and aberrant bone outgrowth(Yucesoy et al.,2015;Hussain et al.,2016).OA is one of the leading causes of cartilage damage.Patients with severe cartilage damage require transplantation of articular cartilage to improve their quality of life.Type Ⅱ collagen is a major component of articular cartilage and intervertebral discs and plays an important role in the structure and strength of connective tissues that support muscles and joints(Byers,1994).

Identifying MicroRNA and mRNA Expression Profiles in Embryonic Stem Cells Derived from Parthenogenetic,Androgenetic and Fertilized Blastocysts

MicroRNAs（miRNAs） are a class of highly conserved small non-coding RNA molecules that play a pivotal role in several cellular functions.In this study,miRNA and messenger RNA（mRNA） profiles were examined by Illumina microarray in mouse embryonic stem cells（ESCs） derived from parthenogenetic,androgenetic,and fertilized blastocysts.The global analysis of miRNA-mRNA target pairs provided insight into the role of miRNAs in gene expression.Results showed that a total of 125 miRNAs and 2394 mRNAs were differentially expressed between androgenetic ESCs（aESCs） and fertilized ESCs（fESCs）,a total of 42 miRNAs and 87 mRNAs were differentially expressed between parthenogenetic ESCs（pESCs） and fESCs,and a total of 99 miRNAs and 1788 mRNAs were differentially expressed between aESCs and pESCs.In addition,a total of 575,5 and 376 miRNA-mRNA target pairs were observed in aESCs vs.fESCs,pESCs vs.fESCs,and aESCs vs.pESCs,respectively.Furthermore,15 known imprinted genes and 16 putative uniparentally expressed miRNAs with high expression levels were confirmed by both microarray and real-time RT-PCR.Finally,transfection of miRNA inhibitors was performed to validate the regulatory relationship between putative maternally expressed miRNAs and target mRNAs. Inhibition of miR-880 increased the expression of Peg3,Dyrklb,and Prrg2 mRNA,inhibition of miR-363 increased the expression of Nfat5 and Soatl mRNA,and inhibition of miR-883b-5p increased Nfat5,Tacstd2,and Ppapdc1 mRNA.These results warrant a functional study to fully understand the underlying regulation of genomic imprinting in early embryo development.

Production of Transgenic Korean Native Cattle Expressing Enhanced Green Fluorescent Protein Using a FIV-Based Lentiviral Vector Injected into MII Oocytes

The potential benefits of generating and using transgenic cattle range from improvements in agriculture to the production of large quantities of pharmaceutically relevant proteins. Previous studies have attempted to produce transgenic cattle and other livestock by pronuclear injection and somatic cell nuclear transfer, but these approaches have been largely ineffective; however, a third approach, lentivirus-mediated transgenesis, has successfully produced transgenic livestock. In this study, we generated transgenic （TG） Korean native cattle using perivitelline space injection of viral vectors, which expressed enhanced green fluorescent protein （EGFP） systemically. Two different types of lentiviral vectors derived from feline immunodeficiency virus （FIV） and human immunodeficiency virus （HIV） carrying EGFP were injected into the perivitelline space of MII oocytes. EGFP expression at 8-cell stage was significantly higher in the FIV group compared to the HIV group （47.5% ± 2.2% v.s. 22.9% 4± 2.9%）. Eight-cell embryos that expressed EGFP were cultured into blastocysts and then transferred into 40 heifers. Ten heifers were successfully impregnated and delivered 10 healthy calves. All of these calves expressed EGFP as detected by in vivo imaging, PCR and Southern blotting. In addition, we established an EGFP-expressing cell line from TG calves, which was followed by nuclear transfer （NT）. Recloned 8-cell embryos also expressed EGFP, and there were no differences in the rates of fusion, cleavage and development between cells derived from TG and non-TG calves, which were subsequently used for NT. These results illustrate that FIV-based lentiviruses are useful for the production of TG cattle. Moreover, our established EGFP cell line can be used for additional studies that involve induced pluripotent stem cells.