Editing MC1R in human melanoma cells by CRISPR/Cas9 and functional analysis.

MC1R (melanocortin 1 receptor) encodes the melanocortin-1 receptor, which can activate intracellular cAMP synthesis under the stimulation of the α-melanocyte stimulating hormone (α-MSH) ligand. Increased cAMP then activates the protein kinase A (PKA) pathway, resulting in the up-regulation of the expression of the microphthalmia-associated transcription factor (MITF) which is a critical regulatory factor of melanin synthesis, and tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis tyrosinase (TYR), and ultimately affects production of eumelanin and pheomelanin, and the coat color phenotype of mammalian species. Previous reports have indicated that the mutation A243T in the transmembrane domain 6 (TM6) of MC1R protein might disrupt the function of MC1R, contributing to the red phenotype in Duroc pig. However, functional analysis of the A243T mutation in MC1R has not yet been carried out. In this study, we attempted to used single-stranded oligo-deoxyribonucleotides (ssODN) as donor templates to introduce the c.727G>A (A243T) mutation into MC1R in human melanoma cell line SK-MEL-2 by CRISPR/Cas9 to analyze its effects on MC1R functions. We found the occurrence of ssODN recombination reached to 10%. Unfortunately, Sanger sequencing MC1R in six single-cell clones revealed that none carried the c.727G>A mutation, but all carried undesired mutations surrounding the target site. Cells transfected with CRISPR/Cas9 plasmids and ssODN presented significantly attenuated cAMP activation, and down-regulated MITF and TYR expression, indicating that the editing MC1R could affect the melanin synthesis function in cells. This study provides a basis for further investigation the mechanism of MC1R mutation on animal coat color.

[Generation of cell strains containing point mutations in HPRT1 by CRISPR/Cas9].

Mutations in Hypoxanthine-guanine Phosphoribosyltransferase1 (HPRT1) gene can lead to metabolic disorder of hypoxanthine and guanine metabolism, and other severe symptoms such as hypophrenia, gout, and kidney stones, called the Lesch-Nyhan disease (LND). Although the mutations are widely distributed throughout the HPRT1 gene, there are some isolated hot spots. In this study, we aim to introduce two previously reported hot spots, c.508 C>T and c.151 C>T, which could lead to premature translational termination in HPRT1 gene. Through CRISPR/Cas9 mediated homology-directed repair (HDR) by using single-stranded oligo-deoxyribonucleotides (ssODN) as donor template, we obtained cell clones containing these two mutations in HEK293T or HeLa cells. Targeted mutation of c.508 C>T and c.151 C>T reached to 16.3% and 10%, respectively. We further detect HPRT1 protein levels with Western blot and enzyme activity with 6-TG in 5 different cell clones. HPRT1 protein and its enzymatic activity both was hardly detected in homozygous mutant cells, while reduced HPRT1 protein expression and enzymatic activity was detected in heterozygous mutant cells. Our study will be beneficial to those who working on generation of cell or animal models of HRPT1 mutations, and provides a basis for further investigations on the genetic mechanism of Lesch-Nyhan disease.

Improving gene targeting efficiency on the porcine BMP15 gene mediated by CRISPR/Cas9 by using the RGS surrogate reporter system.

As Chinese have raised most pigs and consumed most pig products in the world, improving the fertility of sow is of economic benefits to the pig industry in China. The sheep BMP15 (bone morphogenetic protein 15) gene has been identified as a major gene for controlling ovulation rates and prolific traits, which are key factors affecting the fertility of livestock. As similar natural occurring mutations in the porcine BMP15 gene have not yet been reported, we speculated that introducing the same prolific sheep mutations into the porcine BMP15 gene by using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system.

Knockdown of CTRP6 inhibits adipogenesis via lipogenic marker genes and Erk1/2 signalling pathway.

C1q/tumor necrosis factor-related protein 6 (CTRP6), an adipose-tissue secretory factor, plays an important role in inflammatory reaction and carcinogenesis. However, the biological function of CTRP6 in adipogenesis remains unclear. In this study, we examined the effects of CTRP6 knockdown on lipogenesis of 3T3-L1 adipocytes. The results showed that after 3T3-L1 adipocytes transfected with anti-CTRP6 small interfering RNA (siRNA), not only levels of secreted CTRP6 protein in the culture medium but also the expression level of the CTRP6 protein in the 3T3-L1 adipocytes was significantly reduced (P < 0.01). In addition, the number of lipid droplets in the adipocytes was reduced, as well as the OD values reflecting the fat content being significantly decreased (P < 0.01). Meanwhile the levels of adipogenic markers, including peroxisome proliferator activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), CCAAT/enhancer-binding protein ß (C/EBPß) and adipocyte fatty acid-binding protein 4 (aP2), were decreased after treatment with anti-CTRP6 siRNA, whereas the expression of adipose triglyceride lipase (ATGL) and triacylglycerol hydrolase (TGH) were increased. Furthermore, after transfection, activity of phosphorylated Erk1/2 (p-Erk1/2) was inhibited in the early stage of differentiation, but in terminal differentiation of adipocytes, its activity was activated. Taken together, the results indicate that knockdown of CTRP6 can inhibit adipogenesis of 3T3-L1 adipocytes through lipogenic marker genes and Erk1/2 signaling pathway.

Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system.

IGF2 (Insulin-like growth factor 2) is a major growth factor affecting porcine fetal and postnatal development. We propose that the precise modification of IGF2 gene of Chinese indigenous pig breed--Lantang pig by genome editing technology could reduce its backfat thickness, and increase its lean meat content. Here, we tested the genome editing activities of zinc finger nucleases (ZFNs) and CRISPR/Cas9 system on IGF2 gene in the Lantang porcine fetal fibroblasts (PEF). The results indicated that CRISPR/Cas9 presented cutting efficiency up to 9.2%, which was significantly higher than that generated by ZFNs with DNA cutting efficiency lower than 1%. However, even by using CRISPR/Cas9, the relatively lower percentage of genetically modified cells in the transfected population was not satisfied for somatic nuclear transfer (SCNT). Therefore, we used a SSA (Single-strand annealing) reporter system to enrich genetically modified cells induced by ZFN or CRISPR/Cas9. T7 endonuclease I assay revealed that this strategy improved genome editing activity of CRISPR/Cas9 by 5 folds, and was even more effective for improving genome editing efficiency of ZFN.

Identification of the proteins required for fatty acid desaturation in zebrafish (Danio rerio).

Zebrafish Δ-5/Δ-6 fatty acid desaturase (Z-FADS) catalyzes the cascade synthesis of long-chain polyunsaturated fatty acids (PUFAs), thereby playing a pivotal role in several biological processes. In the current study, we report that the Z-FADS protein exists in close proximity to certain cytochrome b5 reductases (CYB5R2 and 3) and elongases (ELOVL2, 4, 5 and 7) on the endoplasmic reticulum, as determined using fluorescence microscopy and fluorescence resonance energy transfer. HeLa cells co-transfected with zebrafish fads and elovl2, 4, and 5 produced docosahexaenoic acid (DHA), as detected by gas chromatography. In addition, immunofluorescence cytochemistry and Western blot data revealed that Z-FADS is present in the mitochondria of HeLa cells. Collectively, our results implicate that Z-FADS, the sole fatty acid desaturase ever been identified in zebrafish, can serve as a universal fatty acid desaturase during lipogenesis.

Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors.

Porcine satellite cells represent an ideal model system for studying the cellular and molecular basis regulating myogenic stem cell proliferation and differentiation and for exploring the experimental conditions for myoblast transplantation. Here, we investigated the effects of mechano growth factor (MGF), a spliced variant of the IGF-1 gene, on porcine satellite cells. We show that MGF potently stimulated proliferation while inhibited differentiation of porcine satellite cells. MGF-treatment acutely down-regulates the expression of myogenic determination factor (MyoD) and the cyclin-dependent kinase inhibitor p21. MGF-treatment also markedly reduced the overall expression of cyclin B1 and key factors of the myogenic regulatory and myocyte enhancer families, including Myogenein and MEF2A. Taken together, the gene expression data from MGF-treated porcine satellite cells are in favor of a molecular model in which MGF inhibits porcine satellite cell differentiation by down-regulating either the activity or expression of MyoD, which, in turn, suppresses the expression of key genes required for cell cycle progression and differentiation, such as p21, Myogenin, and MEF2. Overall, our findings are in support of the previous suggestion that MGF may be used in vivo and in vitro to promote proliferation of myogenic stem cells to prevent and treat age-related muscle degenerative diseases.

[Effects of PI3K/AKT inhibitor wortmannin on proliferation and apoptosis of primary porcine preadipocytes].

The purpose of this study was to determine the proper concentration of wortmannin that effectively inhibits PI3K/AKT but does not affect the proliferation and apoptosis of primary porcine preadipocytes. Firstly, primary porcine preadipocytes were isolated and their abilities to be induced to differentiation into mature adipocytes were evaluated. The preadipocytes were then treated with different concentrations of wortmannin, and the proliferation of the cells was detected with methanethiosulfonate (MTS). Annexin V- FITC/PI double-staining was used to detect the level of cell apoptosis. The apoptosis-related gene expressions were also quantified by qRT-PCR. At the same time, single cell electrophoresis was used to examine the extent of cellular DNA damage. Our data demonstrated that the primary porcine preadipocytes could differ-entiate into mature adipocytes. Up to 200 nmol/L of wortmannin had no effect on the proliferation ability of primary porcine preadipocytes (P>0.05). Results from the flow cytometry Annexin V- FITC/PI double-staining showed that 200 nmol/L wortmannin significantly induced apoptosis of the primary porcine preadipocytes (P<0.05). QRT-PCR results also showed that the expressions of caspase8, TNFR1, GZMB, and Bcl-x1 were significantly upregulated, while the expression of GZMA and cFLIP were not significantly affected when treated with 200 nmol/L wortmannin. In addition, results from the single cell gel electrophoresis indicated that 100 nmol/L wortmannin did not induce DNA damage. In conclusion, our results col-lectively showed that 100 nmol/L wortmanin can be used to study the role of PI3k pathway on the preadipocytes differen-tion without affecting the cell proliferation and apoptosis.

Molecular cloning, expression, and chromosomal mapping of the porcine CDC-2-like kinase 1 (CLK1) gene.

CDC-2-like kinase 1 (CLK1) plays a critical role in regulating pre-RNA splicing and post-transcriptional gene expression. Two distinct transcripts of the porcine CLK1 gene, known as full-length CLK1 and truncated CLK1 (CLK1 ( T )), were identified by in silico cloning, RT-PCR, and RACE. The entire cDNA sequence of full-length CLK1 was 1771 bp, containing a 1455 bp ORF encoding a deduced protein of 484 amino acids. The complete cDNA sequence of CLK1 ( T ) is 1680 bp, containing a 414 bp ORF encoding a deduced protein of 137 amino acids. The genomic structure and sequence of porcine CLK1 were analyzed using a bacterial artificial chromosome clone of a Chinese Erhualian pig, with 13 exons and 12 introns spanning approximately 9 kb. RT-PCR revealed that the full-length and truncated splice forms were expressed at equivalent levels in pig heart, fat, liver, spleen, and lymph tissues. The full-length splice form was expressed at a much higher level than the truncated form in tissues of the pig cerebrum, longissimus dorsi, small intestine, and kidney. The CLK1 gene was physically assigned to SSC 15 between microsatellite markers SW1316 and SW2083 using the IMpRH panels.

[Methylation status of pig adiponectin promoter and its expression].

Adiponectin, a cytokine hormone secreted exclusively by adipose tissue, has key roles in energy homeostasis and in metabolism of glucose and lipid. Adiponectin expression was negatively associated with obesity. Many CpG sites were found at the adiponectin promoter region (nucleotides -1500 approximately -1350 bp). To further understand the regulation of pig adiponectin expression, the methylation status of pig adiponectin promoter and its mRNA expression were analyzed by methylation special PCR (MSP) and real-time PCR. At the adiponectin promoter region where CG enriches (nucleotides -1500 approximately -1350 bp), the percentage of demethylation in Changbai pigs was 83%; and the percentages of demethylation in Lantang pigs at 90-day-old and adult stages were 33% and 100%, respectively. The process of methylation and demethyla-tion mainly occurred in certain CpG sites. In muscle tissues, the promoter hypermethylation status of adiponectin gene was detected, which was consistent with the expression of this gene. These results suggested that the methylation of this gene experienced a dynamic process, with the development of individuals, which agreed with the fluctuating trend of gene expression.

Persistent Supercooled Drizzle at Temperatures below -25°C Observed at McMurdo Station, Antarctica.

The rarity of reports in the literature of brief and spatially limited observations of drizzle at temperatures below -20°C suggest that riming and other temperature-dependent cloud microphysical processes such as heterogeneous ice nucleation and ice crystal depositional growth prevent drizzle persistence in cold environments. In this study, we report on a persistent drizzle event observed by ground-based remote-sensing measurements at McMurdo Station, Antarctica. The temperatures in the drizzle-producing cloud were below -25°C and the drizzle persisted for a period exceeding 7.5 hours. Using ground-based, satellite, and reanalysis data we conclude that drizzle was likely present in parts of a widespread cloud field, which stretched more than ~1000 km along the Ross Ice Shelf coast. Parameter space sensitivity tests using two-moment bulk microphysics in large-eddy simulations constrained by the observations suggest that activated ice freezing nuclei (IFN) and accumulation-mode aerosol number concentrations aloft during this persistent drizzle period were likely on the order of 0.2 L-1 and 20 cm-3, respectively. In such constrained simulations, the drizzle moisture flux through cloud base exceeds that of ice. The simulations also indicate that drizzle can lead to the formation of multiple peaks in cloud water content profiles. This study suggests that persistent drizzle at these low temperatures may be common at the low aerosol concentrations typical of the Antarctic and Southern Ocean atmospheres.

The PmoB subunit of particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath): The CuI sponge and its function.

In this study, we describe efforts to clarify the role of the copper cofactors associated with subunit B (PmoB) of the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) (M. capsulatus). This subunit exhibits strong affinity toward CuI ions. To elucidate the high copper affinity of the subunit, the full-length PmoB, and the N-terminal truncated mutants PmoB33-414 and PmoB55-414, each fused to the maltose-binding protein (MBP), are cloned and over-expressed into Escherichia coli (E. coli) K12 TB1 cells. The Y374F, Y374S and M300L mutants of these protein constructs are also studied. When this E. coli is grown with the pmoB gene in 1.0 mM CuII, it behaves like M. capsulatus (Bath) cultured under high copper stress with abundant membrane accumulation and high CuI content. The recombinant PmoB proteins are verified by Western blotting of antibodies directed against the MBP sub-domain in each of the copper-enriched PmoB proteins. Cu K-edge X-ray absorption near edge spectroscopy (XANES) of the copper ions confirms that all the PmoB recombinants are CuI proteins. All the PmoB proteins show evidence of a "dicopper site" according to analysis of the Cu extended X-ray absorption edge fine structure (EXAFS) of the membranes. No specific activities toward methane and propene oxidation are observed with the recombinant membrane-bound PmoB proteins. However, significant production of hydrogen peroxide is observed in the case of the PmoB33-414 mutant. Reaction of the dicopper site with dioxygen produces hydrogen peroxide and leads to oxidation of the CuI ions residing in the C-terminal sub-domain of the PmoB subunit.

Differential display of grouper iridovirus-infected grouper cells by immunostaining.

Grouper iridovirus (GIV) is one of the most devastating infectious pathogens of aquaculture fish. When infecting a susceptible cell line, such as GK-2, GIV causes antigenic changes in host cellular proteins. To understand the host gene expression characteristics after viral infection, we developed an immunostaining method to screen differentially expressed genes of fish cells in response to GIV infection using phage display complementary DNA libraries. In total, 66 genes were identified from grouper kidney and brain cell lines. These genes are related to replication, transcription, translation, immunity, apoptosis, structure proteins, metabolism, energy, protein modification, and homeostasis. Four dynamic antigenic patterns were observed among these immunocloned genes upon GIV infection. Microarray analysis further confirmed the transcriptional patterns of 80% of the identified genes. This immunostaining screening method provides insights into a host's cellular protein response to viral infection on a translational basis.

Taiwan Biobank: making cross-database convergence possible in the Big Data era.

The Taiwan Biobank (TWB) is a biomedical research database of biopsy data from 200 000 participants. Access to this database has been granted to research communities taking part in the development of precision medicines; however, this has raised issues surrounding TWB's access to electronic medical records (EMRs). The Personal Data Protection Act of Taiwan restricts access to EMRs for purposes not covered by patients' original consent. This commentary explores possible legal solutions to help ensure that the access TWB has to EMR abides with legal obligations, and with governance frameworks associated with ethical, legal, and social implications. We suggest utilizing "hash function" algorithms to create nonretrospective, anonymized data for the purpose of cross-transmission and/or linkage with EMR.

Evaluation of the effect and profitability of gene-assisted selection in pig breeding system.

OBJECTIVE: To evaluate the effect and profitability of using the quantitative trait loci (QTL)-linked direct marker (DR marker) in gene-assisted selection (GAS). METHODS: Three populations (100, 200, or 300 sows plus 10 boars within each group) with segregating QTL were simulated stochastically. Five economic traits were investigated, including number of born alive (NBA), average daily gain to 100 kg body weight (ADG), feed conversion ratio (FCR), back fat at 100 kg body weight (BF) and intramuscular fat (IMF). Selection was based on the estimated breeding value (EBV) of each trait. The starting frequencies of the QTL's favorable allele were 0.1, 0.3 and 0.5, respectively. The economic return was calculated by gene flow method. RESULTS: The selection efficiency was higher than 100% when DR markers were used in GAS for 5 traits. The selection efficiency for NBA was the highest, and the lowest was for ADG whose QTL had the lowest variance. The mixed model applied DR markers and obtained higher extra genetic gain and extra economic returns. We also found that the lower the frequency of the favorable allele of the QTL, the higher the extra return obtained. CONCLUSION: GAS is an effective selection scheme to increase the genetic gain and the economic returns in pig breeding.

[Analysis of microsatellite markers on four chromosomes in F2 design pig resource family].

Lantang pig (16 sows), which is one of the south China type pig breeds, was crossed with Landrace pig (8 boars) to construct the resource population. According to the pig linkage map of USDA-MARC2.0, 31 microsatellite DNA markers on pig chromosomes 1, 4, 7, and 8 were used to genotype the parents F1 and F2. The distance between adjacent markers was about 10 to 20 cM. The gene frequency, heterozygosity (h), and polymorphism information content (PIC) were calculated. The marker genotype of parents, F1, and F2 were obtained with WAVEO nucleotide fragment analysis system (DHPLC) and ABI 377 DNA sequencer. Twenty-one microsatellite markers on chromosomes 1, 4, and 8 were genotyped with ABI 377. The length of the DNA fragments of 18 alleles on 13 microsatellite markers were beyond the range reported on the web site. The loci of new alleles were 62% of total markers. The heterozygosities of the 31 microsatellite ranged from 0.043 to 0.7855, the heterozygosities of 70% loci were over 0.6, the average heterozygosity was 0.6460. The average polymorphism information content (PIC) of 31 microsatellite markers was 0.5949, the PIC of 77.4% loci were over 0.5. The values of h and PIC suggested that polymorphism information of these markers in the resource population was plentiful. These markers could be used to map quantitative traits loci of important economic traits in this population.

Changes in variance components of flanking marker genotypes under varying selection intensities.

Selection is practically ubiquitous during marker-QTL linkage analysis with an experimental population. Thus, it is necessary to investigate the impacts of selection upon linkage analyses in order to obtain unbiased estimates of QTL position and effect. In this article, by exploiting flanking markers through the widely applied half-sib design, we have developed the structures of three variance components, i.e., variance component between marker genotypes, polygenic variance component and recombinant variance component within marker genotypes. Changes in these variance components under varying selection intensities were investigated in this study to formulate the effects of selection on various variance components. Results showed clearly that all variance components presented were quite sensitive to changes in selection intensity. As selection intensity increased, all variance components declined by differing extents in a quadratic fashion. Comparatively speaking, the variance between marker genotypes decreased most drastically, followed by the polygenic variance within marker genotypes and then the recombinant variance within marker genotypes, which suggested a decrease of power for QTL linkage analysis. Therefore, steps should be taken to avoid as much as possible the presence of selection in real populations, so as to further eliminate the negative effects of selection on QTL linkage analysis.

[Cloning and expression of the pig fast skeletal muscle troponin C gene].

A pig skeletal muscle expressed sequence tag (EST, accession number BM083186) was found to encode amino acid sequences with high homology to human TNNC2. In silico cloning obtained a 843 bp sequence which we named pig fast skeletal troponin C gene (TNNC2). The open reading frame of TNNC2 was between nucleotides 201 - 683, and was confirmed by RT-PCR. The gene encoded a 160 amino acid residue protein and had the highest similarity with human and mouse homologues, with percent identities being 93.6%, 90.5%, respectively. Semi-quantitative RT-PCR analysis of mRNA from different normal tissues indicted that the TNNC2 gene was expressed in the skeletal muscle and the levels of expression were higher in the longissimus dorsi of the Duroc pig than in the same tissue of the Lantang pig.

High throughput cytotoxicity screening of anti-HER2 immunotoxins conjugated with antibody fragments from phage-displayed synthetic antibody libraries.

Immunotoxins are an important class of antibody-based therapeutics. The potency of the immunotoxins depends on the antibody fragments as the guiding modules targeting designated molecules on cell surfaces. Phage-displayed synthetic antibody scFv libraries provide abundant antibody fragment candidates as targeting modules for the immunoconjugates, but the discovery of optimally functional immunoconjugates is limited by the scFv-payload conjugation procedure. In this work, cytotoxicity screening of non-covalently assembled immunotoxins was developed in high throughput format to discover highly functional synthetic antibody fragments for delivering toxin payloads. The principles governing the efficiency of the antibodies as targeting modules have been elucidated from large volume of cytotoxicity data: (a) epitope and paratope of the antibody-based targeting module are major determinants for the potency of the immunotoxins; (b) immunotoxins with bivalent antibody-based targeting modules are generally superior in cytotoxic potency to those with corresponding monovalent targeting module; and (c) the potency of the immunotoxins is positively correlated with the densities of the cell surface antigen. These findings suggest that screening against the target cells with a large pool of antibodies from synthetic antibody libraries without the limitations of natural antibody responses can lead to optimal potency and minimal off-target toxicity of the immunoconjugates.

[Effect of population size of performance test on short-term selection result of sire line].

The effects of the population size,boar to sow ratio,and the number of pigs tested per litter on the selection response and its coefficient of variation, inbreeding coefficient of sire line selection were studied. Pig populations of 100 to 500 sows were simulated with Monte Carlo method,and the ratios of boar to sow were 1:10 and 1:20. The number of pigs tested per litter were 2 and 4 composed equally of boars and gilts. The traits selected were average daily gain after weaning (ADG) and backfat thickness (BF) at 100 kg live weight. Breeding pigs were selected according to multiple-trait BLUP,and economic weight of BF was set as 2.5 times of that of ADG. After five generation selection, the results showed that the more the number of sows of the breeding population, the higher the cumulative selection response at 5th generation, and the slower the coefficient of inbreeding increment per generation, the smaller the coefficient of variation of the cumulative selection response at 5th generation. Increasing the number of pigs tested per litter and/or the boar to sow ratio increased the cumulative selection response at 5th generation, inbreeding coefficient increment per generation, and the coefficient of variation of the cumulative selection response at 5th generation. The cumulative selection response got higher and the increment of inbreeding coefficient slowed down significantly when the number of sows in the breeding population increased from 100 to 300,and the ratio of boar to sow and the number of pigs tested per litter were fixed. When the sow number of breeding population was increased from 300 to 400 above,the cumulative selection response only increased slightly,and the coefficient of inbreeding continued to decrease but at a diminished rate. In conclusion, for the short-term selection of sire line, it is recommended that the breeding population should be composed of 400 or above sows, 4 pigs tested per litter,and the ratio of boar to sow should be maintained at 1:20.