ResearchPad - animal-biotechnology https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Histone deacetylases inhibitor and RAD51 recombinase increase transcription activator-like effector nucleases-mediated homologous recombination on the bovine β-casein gene locus]]> https://www.researchpad.co/article/elastic_article_14450 The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system.MethodsMAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site.ResultsThe level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate.ConclusionThese results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells. ]]> <![CDATA[Effect of all-trans retinoic acid on casein and fatty acid synthesis in MAC-T cells]]> https://www.researchpad.co/article/elastic_article_14446 Caseins and fatty acids of milk are synthesized and secreted by the epithelial cells of the mammary gland. All-trans retinoic acid (ATRA), an active metabolite of vitamin A, has been shown to promote mammary development. This study was conducted to determine the effect of ATRA on casein synthesis and fatty acid composition in MAC-T cells.MethodsMAC-T cells were allowed to differentiate for 4 d, treated with ATRA (0, 1.0, 1.5, and 2.0 μM), and incubated for 3 d. We analyzed the fatty acid composition, the mRNA expression of casein and fatty acid synthesis-related genes, and the phosphorylation of casein synthesis-related proteins of MAC-T cells by gas chromatography, quantitative polymerase chain reaction, and western blotting, respectively.ResultsIn MAC-T cells, ATRA increased the mRNA levels of αS1-casein and β-casein, janus kinase 2 (JAK2) and E74-like factor 5 of the signal transducer and activator of transcription 5 β (STAT5-β) pathway, ribosomal protein S6 kinase beta-1 (S6K1) and eukaryotic translation initiation factor 4E binding protein 1 of the mammalian target of rapamycin (mTOR) pathway, inhibited the mRNA expression of phosphoinositide 3-kinase and eukaryotic initiation factor 4E of the mTOR pathway, and promoted the phosphorylation of STAT5-β and S6K1 proteins. Additionally, ATRA increased the de novo synthesis of fatty acids, reduced the content of long-chain fatty acids, the ratio of monounsaturated fatty acids to saturated fatty acids (SFA), the ratio of polyunsaturated fatty acids (PUFA) to SFA, and the ratio of ω-6 to ω-3 PUFA. The mRNA levels of acetyl-CoA carboxylase 1, fatty acid synthase, lipoprotein lipase, stearoyl-CoA desaturase, peroxisome proliferator-activated receptor gamma, and sterol regulatory element-binding protein 1 (SREBP1) were enhanced by ATRA.ConclusionATRA promotes the synthesis of casein by regulating JAK2/STAT5 pathway and downstream mTOR signaling pathway, and it improves the fatty acid composition of MAC-T cells by regulating SREBP1-related genes. ]]> <![CDATA[In vitro and in vivo pharmacokinetic characterization of LMT-28 as a novel small molecular interleukin-6 inhibitor]]> https://www.researchpad.co/article/N6affef90-6610-4ac9-bfb9-6453ed1694a2

Objective

Interleukin-6 (IL-6) is a T cell-derived B cell stimulating factor which plays an important role in inflammatory diseases. In this study, the pharmacokinetic properties of LMT-28 including physicochemical property, in vitro liver microsomal stability and an in vivo pharmacokinetic study using BALB/c mice were characterized.

Methods

LMT-28 has been synthesized and is being developed as a novel therapeutic IL-6 inhibitor. The physicochemical properties and in vitro pharmacokinetic profiles such as liver microsomal stability and Madin-Darby canine kidney (MDCK) cell permeability assay were examined. For in vivo pharmacokinetic studies, pharmacokinetic parameters using BALB/c mice were calculated.

Results

The logarithm of the partition coefficient value (LogP; 3.65) and the apparent permeability coefficient values (Papp; 9.7×10−6 cm/s) showed that LMT-28 possesses a moderate-high cell permeability property across MDCK cell monolayers. The plasma protein binding rate of LMT-28 was 92.4% and mostly bound to serum albumin. The metabolic half-life (t1/2) values of LMT-28 were 15.3 min for rat and 21.9 min for human at the concentration 1 μM. The area under the plasma drug concentration-time curve and Cmax after oral administration (5 mg/kg) of LMT-28 were 302±209 h·ng/mL and 137±100 ng/mL, respectively.

Conclusion

These data suggest that LMT-28 may have good physicochemical and pharmacokinetic properties and may be a novel oral drug candidate as the first synthetic IL-6 inhibitor to ameliorate mammalian inflammation.

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<![CDATA[Transdifferentiation of bovine epithelial cells towards adipocytes in the presence of myoepithelium]]> https://www.researchpad.co/article/N84397d0c-d13e-451d-8eef-35c98df943e5

Objective

Orchastric changes in the mammary glands are vital, especially during lactation. The secretary epithelial cells together with the supporting myoepithelial and stromal cells function cordially to secrete milk. Increase in the number of luminal epithelial cells and a decrease in adipocytes are visible during lactation, whereas the reverse happens in the involution. However, an early involution occurs if the epithelial cells transdifferentiate towards adipocytes during the lactation period. We aimed to inhibit the adipocyte transdifferentiation of luminal cells by restraining the peroxisomal proliferator-activated receptor γ (PPARγ) pathway.

Methods

Linolenic acid (LA) and thiazolidinediones (TZDs) induced adipogenesis in mammary epithelial cells were conducted in monolayer, mixed culture as well as in transwell plate co-culture with mammary myoepithelial cells.

Results

Co-culture with myoepithelial cells showed higher adipogenic gene expression in epithelial cells under LA+TZDs treatment. Increase in the expressions of PPARγ, CCAAT/enhancer-binding protein α and vimentin in both mRNA as well as protein levels were observed. Whereas, bisphenol A diglycidyl ether treatment blocked LA+TZDs induced adipogenesis, as it could not show a significant rise in adipose related markers. Although comparative results were found in both mixed culture and monolayer conditions, co-culture technic was found to work better than the others.

Conclusion

Antagonizing PPARγ pathway in the presence of myoepithelial cells can significantly reduce the adipogenisis in epithelial cells, suggesting therapeutic inhibition of PPARγ can be considered to counter early involution or excessive adipogenesis in mammary epithelium in animals.

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<![CDATA[No excessive mutations in transcription activator-like effector nuclease-mediated α-1,3-galactosyltransferase knockout Yucatan miniature pigs]]> https://www.researchpad.co/article/N68b8815b-3ffa-4fdc-a6f6-413c4bb4111e

Objective

Specific genomic sites can be recognized and permanently modified by genome editing. The discovery of endonucleases has advanced genome editing in pigs, attenuating xenograft rejection and cross-species disease transmission. However, off-target mutagenesis caused by these nucleases is a major barrier to putative clinical applications. Furthermore, off-target mutagenesis by genome editing has not yet been addressed in pigs.

Methods

Here, we generated genetically inheritable α-1,3-galactosyltransferase (GGTA1) knockout Yucatan miniature pigs by combining transcription activator-like effector nuclease (TALEN) and nuclear transfer. For precise estimation of genomic mutations induced by TALEN in GGTA1 knockout pigs, we obtained the whole-genome sequence of the donor cells for use as an internal control genome.

Results

In-depth whole-genome sequencing analysis demonstrated that TALEN-mediated GGTA1 knockout pigs had a comparable mutation rate to homologous recombination-treated pigs and wild-type strain controls. RNA sequencing analysis associated with genomic mutations revealed that TALEN-induced off-target mutations had no discernable effect on RNA transcript abundance.

Conclusion

Therefore, TALEN appears to be a precise and safe tool for generating genome-edited pigs, and the TALEN-mediated GGTA1 knockout Yucatan miniature pigs produced in this study can serve as a safe and effective organ and tissue resource for clinical applications.

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<![CDATA[Detection of myostatin gene MSTN in some goat breeds (Capra hircus)]]> https://www.researchpad.co/article/5c6b03c7d5eed0c484267777

Till now not information about myostatin MSTN gene in Egyptian goat breeds. Here we show more information about MSTN in some Egyptian goat breeds to enrich the database with new sequences for Egyptian goat breeds. Our conducted study focused on detection and identifying the MSTN gene as a candidate gene of the muscles growth trait in three goat breeds (Zaraibi, Baladi and Damascus). We found the similarity between the registered sequences with the accession numbers KY463684 for Zaraibi and KY463685 for Baladi and Chinese goat breeds of the MSTN gene deposited with international gene banks by up to 99% and some other species including sheep, cows and bull breeds with percentages of 95 to 97% and between 95 to 99%, respectively. There is also a correlation between the sequences of the registered pieces of Baladi with KY463686 and Damascus and Chinese breeds with KY441464 of MSTN deposited with international gene banks by up to 99% and some other species including sheep and bull breeds at a ratio of 99% for two pieces. Results demonstrated the deposited sequences of object are part of intron 1, exon 2 is fully sequenced with Zaraibi and Baladi breeds; the intron 1, exon 1 with Baladi breed; and the intron 2, part of exon 3 with Damascus breed. Therefore, the Egyptian goat breeds consider national wealth can be used to develop breeding and improvement programs which helps in more applicable scopes like biotechnology, genetic engineering and molecular biology with the help of bioinformatics tools.

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<![CDATA[Buffalo species identification and delineation using genetic barcoding markers]]> https://www.researchpad.co/article/5c6b03e9d5eed0c484267940

Enrichment of barcode databases with mitochondrial cytochrome c oxidase subunit I (COI) barcode sequences in different animal taxa has become important for identification of animal source in food samples to prevent commercial fraud. In this study, COI barcode sequence in seventy one river buffalo samples were determined, analyzed and deposited in Genbank barcode database and barcode of life database (BOLD) to contribute for construction of public reference library for COI barcode sequence in river buffalo. Moreover COI barcode sequence was used to identify the closely related buffalo groups: river buffalo, swamp buffalo, lowland anoa and African buffalo. Results indicated the success of the COI barcode in the identification of each of the tested groups. Whereas a suggested sequence of other mitochondrial segment representing two successive transfer RNA (tRNA) genes; tRNA-Threonine (MT-TT) and tRNA-Proline (MT-TP) was failed to be used as a barcode marker for differentiation between the tested buffalo groups.

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<![CDATA[Five BoLA-DRB3 genotypes detected in Egyptian buffalo infected with Foot and Mouth disease virus serotype O]]> https://www.researchpad.co/article/5c6b03b3d5eed0c4842675e3

Foot and Mouth disease (FMD) is a contagious disease leads to economically loss in livestock production all over the world. This serious disease is caused due to the infection of the animal with a single-stranded RNA virus (FMDV). This study aimed to investigate the genetic polymorphism of BoLA-DRB3 gene in Egyptian buffalo as a candidate genetic marker included in multi-factorial process of FMD resistance/susceptibility. Also this work aimed to genetically characterization and serotyping of circulating FMD virus in Egypt during 2016.

For serotyping of FMDV, RT-PCR was used for FMDV-positive samples and the results declared the presence of serotype O in all tested animals. The sequence analysis of FMDV samples revealed five different patterns for the detected serotype O which were submitted to GenBank under the accession Nos.: MG017361–MG017365.

The 302-bp amplified fragments from BoLA-DRB3 exon 2 were digested with HaeIII endonuclease and the results showed that the presence of five BoLA-DRB3 genotypes, among them the genotype AA might be associated with FMD-resistance (P < 0.01). On the other hand, genotype AC could be correlated with susceptibility (P < 0.01) to FMD in Egyptian buffaloes where it was absent in resistant group. The five detected genotypes of BoLA-DRB3 exon 2 were submitted to GenBank with the accession Nos.: MF977316–MF977320. In conclusion, our findings suggested that the detection of different BoLA-DRB3 genotypes may be has a promising role for raising the resistance of Egyptian buffalo against FMDV especially serotype O which is prevalent in Egypt with preferring genotype AA.

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<![CDATA[Feline panleukopenia viral infection in cats: Application of some molecular methods used for its diagnosis]]> https://www.researchpad.co/article/5c6b03e7d5eed0c484267932

Forty diseased cats and seven healthy control cats from different sex, ages and breeds had examined clinically to confirm presence or absence of clinical symptoms of Feline panleukopenia disease (FP). Several tools including ELISA, gene expression analysis (qRT-PCR), DNA fragmentation test and apoptosis assay were conducted to determine the Feline panleukopenia disease in cat tissues. Clinical symptoms in the form of depression, fever, anorexia, vomiting, diarrhea, dehydration, anaemia and leucopenia were recorded in the diseased cats while no clinical sings were observed in control healthy cats. ELISA results showed that all of diseased (n = 40) cats were positive while control cats (n = 7) were negative for FP viral antigen. After carrying out of ELISA assay, supportive treatment trials including fluid therapy, immunostimulant, antibiotics to overcome dehydration, restoring electrolytes imbalances, combating secondary bacterial infection were conducted but all diseased cats were died and control cats exposed to soft death. Gene expression analysis detected high levels of FP viral gene in several cat tissues in which ilium exhibited high viral expression levels compared with jejunum. Also, viral expression levels in jejunum were higher than in mesenteric lymph nodes. In addition, viral expression levels were not detected in tissues of control cats. The results of the DNA fragmentation assay observed that DNA extracted from different tissues of infected cats exhibited damaged DNA bands as compared with DNA of control cats. DNA fragmentation rates in infected tissues increased significantly (P < 0.01), the highest rates were showed in ilium and jejunum tissue than in mesenteric lymph nodes. Determination of apoptosis in cat tissues showed that rate of apoptosis/necrosis increased significantly (P < 0.05) in infected cats tissues in comparison to control cats. Moreover the highest apoptotic ratios of infected cats were observed in ilium and jejunum tissues compared with mesenteric lymph nodes.

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<![CDATA[Genome analysis of Yucatan miniature pigs to assess their potential as biomedical model animals]]> https://www.researchpad.co/article/5c63704cd5eed0c484b29d81

Objective

Pigs share many physiological, anatomical and genomic similarities with humans, which make them suitable models for biomedical researches. Understanding the genetic status of Yucatan miniature pigs (YMPs) and their association with human diseases will help to assess their potential as biomedical model animals. This study was performed to identify non-synonymous single nucleotide polymorphisms (nsSNPs) in selective sweep regions of the genome of YMPs and present the genetic nsSNP distributions that are potentially associated with disease occurrence in humans.

Methods

nsSNPs in whole genome resequencing data from 12 YMPs were identified and annotated to predict their possible effects on protein function. Sorting intolerant from tolerant (SIFT) and polymorphism phenotyping v2 analyses were used, and gene ontology (GO) network and Kyoto encyclopedia of genes and genomes (KEGG) pathway analyses were performed.

Results

The results showed that 8,462 genes, encompassing 72,067 nsSNPs were identified, and 118 nsSNPs in 46 genes were predicted as deleterious. GO network analysis classified 13 genes into 5 GO terms (p<0.05) that were associated with kidney development and metabolic processes. Seven genes encompassing nsSNPs were classified into the term associated with Alzheimer’s disease by referencing the genetic association database. The KEGG pathway analysis identified only one significantly enriched pathway (p<0.05), hsa04080: Neuroactive ligand-receptor interaction, among the transcripts.

Conclusion

The number of deleterious nsSNPs in YMPs was identified and then these variants-containing genes in YMPs data were adopted as the putative human diseases-related genes. The results revealed that many genes encompassing nsSNPs in YMPs were related to the various human genes which are potentially associated with kidney development and metabolic processes as well as human disease occurrence.

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<![CDATA[Cytochrome b conservation between six camel breeds reared in Egypt]]> https://www.researchpad.co/article/5c47a614d5eed0c484c742b2

This study was aimed to assess cytochrome b conservation in six breeds of camels reared in Egypt and to compare its sequence with those of other livestock species. The 208-bp fragments from camel mtDNA cyto b were amplified using PCR for 54 camels belonging to 6 camel breeds reared in Egypt. The alignment of camel cyto b sequences showed the presence of two polymorphic sites resulting in four haplotypes and their nucleotide sequences were submitted to GenBank under the accession numbers: KX909894–KX909897.

The genetic distances between tested camel breeds were zero between Baladi, Fallahi and Maghrabi breeds whereas they were at low value between the other three breeds: Mowaled, Sodany and Somali. Neighbor-joining showed 4 branches; one of them include most of the tested animals and another one contains 2 Somali animals which is considered a specific haplotype for this breed. The other two branches are mixed between Sodani and Mowaled breeds.

Neighbor-joining tree was constructed between cyto b sequences of our tested camels and their sequences from livestock species include Camelus dromedaries, Camelus bactrianus, Ovis aries, Capra hircus, Bubalus bubalis, Bos Taurus and Sus scrofa. The result confirmed that our camel breeds belong to Camelus dromedaries and are clearly separated from other species.

It is concluded that cyto b sequence is highly conserved among all camel breeds reared in Egypt which belong to Camelus dromedaries in addition to the advantage of cyto b in differentiation between different livestock sources which enables it to widely use for the adulteration detection in mixed meat.

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<![CDATA[Phylogenetic analysis of Aceh cattle breed of Indonesia through mitochondrial D-Loop region]]> https://www.researchpad.co/article/5c47a6f8d5eed0c484c79bc5

The objective of this research was to find the basic data on genetic diversity of mtDNA D-Loop in Aceh cattle and its association with Bhutanese, Chinese, and Indian cattle. There were sixty samples of DNA which had been sequenced; i.e. Banda Aceh (11), Saree (20), and Indrapuri (29). To the best of our knowledge this is the first published data on the complete mitochondrial D-Loop sequence of Aceh cattle. Results show that Aceh cattle have the closest relationship to Bos indicus and have been influenced by Bos taurus. The closest genetic ranges among Aceh cattle, Bhutanese, Chinese, Indian and Zebu were Aceh–Zebu (0.0138), Aceh–Bhutanese (0.0156), Aceh–Chinese (0.0190) and Aceh–Indian (0.0193). D-Loop mtDNA analyses showed that there were 27 haplotypes in which twenty-one samples spread in haplotype 1, two samples were in haplotype 2, and the other four haplotypes had various samples in the range of three to seventeen samples. One sample of Aceh cattle from Saree has a closest maternal genetic with B. taurus. One of the four mutations among the star-shaped clusters on median joining network was a new specific haploid-group in Aceh cattle. From this finding it could be assumed that Aceh cattle form a specific haplotype and it can be conclude that Aceh cattle are animal genetic resources from Aceh in Sumatera Island that have to be preserved.

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<![CDATA[Effects of age variance on repeatability estimates of egg dimensions of Bovan Nera Black laying chickens]]> https://www.researchpad.co/article/5c47a6cad5eed0c484c78a1f

The present research was designed to examine the effects of age variance on repeatability estimates of egg length, egg breadth and egg shape index of Bovan Nera Black laying chickens at 25, 51, 72 weeks and combined ages of the bird. For this purpose thirty birds were selected from the flock of layers in the Babcock University Teaching and Research Farm. They were individually housed in labeled separate battery cage. A total of thirty (30) eggs were collected daily from the birds continuously for five (5) days of egg production, at each age of 25, 51 and 72 weeks. The total number of eggs collected at each age were 150 and 450 for the total of three age periods. Data were collected on egg production traits for egg length, egg breadth and egg shape index. These data were subjected to statistical analysis using Completely Randomized Design. General linear model procedure of statistical analytical system (SAS) was used to obtain the variance components for the estimation of repeatability. Moderate repeatability estimates were obtained when the age variance was included in the computation and low estimates were registered when the age variance was excluded from the computation. The repeatability estimates from different egg quality traits were low to high. Since most of the traits recorded low repeatability values, these traits can be improve by mass selection thereby culminating into egg production with optimal quality.

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