Establishment and verification of a real-time quantitative PCR method for determination of exogenous antibody gene copy number in CHO cells / 中国生物制品学杂志

@#Objective To establish a real-time quantitative PCR method using SYBR GreenⅠto detect the copy numbers of light chain(LC)and heavy chain(HC)of exogenous antibody gene in CHO cells,and verify and preliminarily apply this method.Methods With the B2m(β2-microglobulin)expressed stably in CHO cells as the internal reference gene,suitable primers of LC,HC genes and internal reference gene were designed respectively,and the reaction system and program of the real-time quantitative PCR method were determined. The established method was verified for the specificity,linearity,precision and durability,and used to detect the copy numbers of LC and HC genes in the recombinant cell lines of working cell bank(WCB)and cells of different passages.Results The primers of exogenous genes and internal reference gene showed specific binding to the target fragments;The efficiency of primer amplification for the B2m gene,LC gene,and HC gene was 106. 7%,106. 3% and 99. 1%,respectively,and the correlation coefficients of the linear equations were all greater than 0. 99 with a good linear relationship;The relative standard deviations(RSDs)of precision verification were all less than 1%;Few cycles of freeze-thaw in a short period had little effect on the detection results. The copy numbers of LC and HC genes in different generations of recombinant cell lines detected by the established method showed no obvious changes.Conclusion A real-time quantitative PCR method for the determination of the copy number of exogenous genes in CHO cells was successfully established with good specificity,linearity,precision and durability,which provides a reference for detecting the copy number of exogenous genes expressed in other CHO cell lines

Impact of C4, C4A and C4B gene copy number variation in the susceptibility, phenotype and progression of systemic lupus erythematosus

Abstract Background Complement component 4 (C4) gene copy number (GCN) affects the susceptibility to systemic lupus erythematosus (SLE) in different populations, however the possible phenotype significance remains to be determined. This study aimed to associate C4A , C4B and total C4 GCN and SLE, focusing on the clinical phenotype and disease progression. Methods C4 , C4A and C4B GCN were determined by real-time PCR in 427 SLE patients and 301 healthy controls, which underwent a detailed clinical evaluation according to a pre-established protocol. Results The risk of developing SLE was 2.62 times higher in subjects with low total C4 GCN (< 4 copies, OR = 2.62, CI = 1.77 to 3.87, p < 0.001) and 3.59 times higher in subjects with low C4A GCN (< 2 copies; OR = 3.59, CI = 2.15 to 5.99, p < 0.001) compared to those subjects with normal or high GCN of total C4 (≥4) and C4A (≥2), respectively. An increased risk was also observed regarding low C4B GCN, albeit to a lesser degree (OR = 1.46, CI = 1.03 to 2.08, p = 0.03). Furthermore, subjects with low C4A GCN had higher permanent disease damage as assessed by the Systemic Lupus International Collaborating Clinics - Damage Index (SLICC-DI; median = 1.5, 95% CI = 1.2-1.9) than patients with normal or high copy number of C4A (median = 1.0, 95% CI = 0.8-1.1; p = 0.004). There was a negative association between low C4A GCN and serositis ( p = 0.02) as well as between low C4B GCN and arthritis ( p = 0.02). Conclusions This study confirms the association between low C4 GCN and SLE susceptibility, and originally demonstrates an association between low C4A GCN and disease severity.

Polymorphic patterns of pfcrt and pfmdr1 in Plasmodium falciparum isolates along the Thai-Myanmar border

Objective: To investigate the distribution and patterns of pfcrt and pfmdr1 polymorphisms in Plasmodium falciparum (P. falciparum) isolates collected from the malaria endemic area of Thailand along Thai-Myanmar border. Methods: Dried blood spot samples were collected from 172 falciparum malaria patients prior received treatment. The samples were extracted using chelex to obtain parasite DNA. PCR-RFLP was employed to detect pfcrt mutation at codons 76, 220, 271, 326, 356 and 371, and the pfmdr1 mutation at codon 86. Pfmdr1 gene copy number was determined by SYBR Green I real-time PCR. Results:Mutant alleles of pfcrt and wild type allele of pfmdr1 were found in almost all samples. Pfmdr1 gene copy number in isolates collected from all areas ranged from 1.0 to 5.0 copies and proportion of isolates carrying>1 gene copies was 38.1%. The distribution and patterns of pfcrt and pfmdr1 mutations were similar in P. falciparum isolates from all areas. However, significant differences in both number of pfmdr1 copies and prevalence of isolates carrying>1 gene copies were observed among isolates collected from different areas. The median pfmdr1 copy number in P. falciparum collected from Kanchanaburi and Mae Hongson were 2.5 and 2.0, respectively and more than half of the isolates carried>1 gene copies.

Association of TLR7 gene copy number variations and systemic lupus erythematosus in Han population / 中华风湿病学杂志

Objective To examine the variation in TLR7 gene copy number of patients with systemic lupus erythematosus (SLE) in Han population,and investigate the relationship between TLR7 gene copy number variations and clinical phenotypes of SLE.Methods Determination of gene copy number of TLR7 was achieved by AccuCopyTM multiple gene copy number detection method in 337 cases of Han SLE patients and 338 healthy controls.According to the clinical phenotype stratification,all cases were divided into lupus nephritis and non-lupus nephritis group,the hematological involvement and non-hematological involvement group,anti-Smith antibody positive and negative group.The data were analyzed by non-parametric rank test.Results Based on sex,there was no significant difference in the variations in TLR7 gene copy number between in female SLE patients and female healthy controls (Z=-1.175,P=0.240).There was also no difference in male group (Z=-1.085,P=0.278).Comparing gene copy numbers variation based on the presence or absence of lupus nephritis,hematological involvement,and anti-Smith antibody,there was no statistical significant difference in female SLE patients(Z=-0.888,P=0.375; Z=-1.085,P=0.278; Z=-0.529,P=0.597).There was no difference in variation in male SLE patients,neither (Z=-0.460,P=0.646; Z=-0.340,P=0.733;Z=-0.158,P=0.874).Conclusion The variations in TLR7 gene copy number are not correlated with SLE and clinical phenotypes of SLE in Han population.