Challenges for the Characterization of Genetically Modified Animals by the qPCR Technique in the Era of Genomic Editing

Characterization of genetically modified organisms through determina­tion of zygosity and transgene integration concerning both copy number and genome site is important for breeding a transgenic line and the use of these organisms in the purpose for which it was obtained.Southern blot,fluorescence in situ hybridization or mating are demanding and time-consuming techniques traditionally used in the characterization of transgenic organisms and,with the exception of mating,give ambiguous results.With the emergence of the real-time quantitative PCR technology,different applications have been described for the analysis of transgenic organisms by determination of several parameters to transgenic analysis.However,the accuracy in quantitation by this method can be influenced in all steps of analysis.This review focuses on the aspects that influence pre-analytical steps(DNA extraction and DNA quantification methods),quantification strategies and data analysis in quantification of copy num­ber and zygosity in transgenic animals.

Depletion of MRPL35 inhibits gastric carcinoma cell proliferation by regulating downstream signaling proteins

BACKGROUND Gastric carcinoma(GC)is a digestive system disease with high morbidity and mortality.However,early clinical detection is difficult,and the therapeutic effect for advanced disease is not satisfactory.Thus,finding new tumor markers and therapeutic targets conducive to the treatment of GC is imperative.MRPL35 is a member of the large subunit family of mitochondrial ribosomal protein.MRPL35 shows the characteristic of oncogene in colorectal cancer and esophageal cancer,which promotes the exploration of the correlation between MRPL35 and GC.We proposed that the expression of MRPL35 might be critical in GC.AIM To study the effect of MRPL35 knockdown on GC cell proliferation.METHODS The expression of MRPL35 in GC was evaluated based on data from the publictumor database UALCAN(www.ualcan.path.uab.edu).The effect of theexpression of MRPL35 on the prognosis was evaluated with KMplot(www.kmplot.com).The expression of MRPL35 was assessed on the tissuemicroarray by immunohistochemistry and the level of MRPL35 mRNA in 25 pairsof clinical GC tissues and matched adjacent tissues was detected by quantitativereverse transcription-polymerase chain reaction.Celigo cell count assay,colonyformation assay,and flow cytometry were used to assess the role of MRPL35 inGC cell proliferation and apoptosis in vitro.Additionally,tumor formationexperiment in BALB/c nude mice was utilized to determine the effect of MRPL35on GC cell proliferation.After knockdown of MRPL35,related proteins wereidentified by isobaric tags for relative and absolute quantification analysis,andthe expression of related proteins was detected by Western blot.RESULTSThe expression of MRPL35 was up-regulated in GC(P=1.77×10^(-4)).The Kaplan-Meier plots of the overall survival indicated that high expression of MRPL35 wasassociated with a poor survival in GC.Compared with adjacent tissues,theexpression of MRPL35 in GC tissues was increased,which was related to age(P=0.03),lymph node metastasis(P=0.007),and pathological tumor-node-metastasisstage(P=0.024).Knockdown of MRPL35 inhibited GC cell proliferation andcolony formation and induced apoptosis.Animal experiment results showed thatknockdown of MRPL35 inhibited tumor formation in BALB/c nude mice.Westernblotting analysis showed that after knockdown of MRPL35,the expression ofPICK1 and BCL-XL proteins decreased,and that of AGR2 protein increased.CONCLUSIONCollectively,our findings demonstrate that knockdown of MRPL35 inhibits GCcell proliferation through related proteins including PICK1,BCL-XL,and AGR2.

iTRAQ-based quantitative proteome characterization of wheat grains during filling stages

Using isobaric tags for relative and absolute quantification （iTRAQ） and associated analytic technologies, we have cataloged and compared 7 069 unique wheat proteins expressed during four substages of the filling stage. Among them, 859 are differentially expressed, showing at least a 2-fold difference in concentration across substages. Differentially expressed proteins （DEPs） includind high-molecular weight giutenin subunit （W5AIU1）, low-molecular weight glutenin subunit （QSW3V4）, gliadin/avenin-like seed protein （D2KFG9）, and avenin-like protein （W5DVL2）, all of which have previously been identified as important for nutritional quality and bread-making properties, and all of which were found to increase at the latter stages of development. We have applied statistical techniques to group the proteins into hierarchical clusters, and have consulted databases to infer functional and other relationships among the identified proteins.

Effect of Drought Stress on Proteome of Maize Grain during Grain Filling

Based on isobaric tags for relative and absolute quantification(iTRAQ)technology,the proteome of grains of a maize cultivar Huangzao 4 under drought stress at grain filling stage was analyzed.The results show that under drought stress,438 proteins were differentially expressed in the maize grains during grain filling.Among them,200 were up-regulated and 238 were down-regulated.The gene ontology(GO)analysis shows that the biological processes in which differential proteins are more involved are cellular processes,metabolic processes and single biological processes;proteins in the cell component category are mainly distributed in cells,cell parts and organelles;and the proteins the molecular function category mainly possess catalytic activity and binding function.Differentially expressed proteins classified by COG are mainly involved in protein post-translational modification and transport,molecular chaperones,general functional genes,translation,ribosomal structure,biosynthesis,energy production and transformation,carbohydrate transport and metabolism,amino acid transport and metabolism,etc.The subcellular structure of the differentially expressed proteins is mainly located in the cell chloroplast and cytosol.The proportions are 35.01%and 30.21%respectively.KEGG metabolic pathway enrichment analysis shows that the differentially expressed proteins are mostly involved in antibiotic biosynthesis,microbial metabolism in different environments,and endoplasmic reticulum protein processing;the metabolic pathways with higher enrichment are the carbon fixation pathway and estrogen signaling pathway of prokaryotes;and the higher enrichment and greater significance are in the tricarboxylic acid cycle,carbon fixation of photosynthetic organisms and proteasome.The results of this study preliminarily reveal the adaptive mechanism of maize grains in response to drought stress during grain filling,providing a theoretical reference for maize drought-resistant molecular breeding.

Quantitative analysis of serum protein in patients with acute coronary syndrome

Background The early diagnosis of acute coronary syndromes(ACS)is of great significance for saving patient′s life. The high-resolution and high-throughput proteomic techniques provide a new means for the discovery of disease-specific biomarkers.We screen serum protein molecules associated with ACS using differential proteomics methods with isotope-labeled relative and absolute quantification(iTRAQ). Method Coronary sinus serum(10 in each group)was collected from ACS patients and healthy controls with the same Genetic background. iTRAQ analysis was performed to screen for significantly different proteins between ACS and healthy controls after pretreatment with high-abundance protein. Result In the iTRAQ analysis of the serum samples from two groups,92 proteins with a mean difference of more than 1.5 times were screened in serum of patients with ACS compared with healthy control serum. The functions of these proteins are involved in stress response,transport,immune response and signal transduction. Conclusions ACS is associated with altered expression of many proteins which may serve as the potential markers of ACS in serum.