Objective: To establish a method of non-isotope double in situ hybridization in order to detect the expression of two kinds of oncogenes at single cell level simultaneously, and confirm the hypothesis of 'model of...Objective: To establish a method of non-isotope double in situ hybridization in order to detect the expression of two kinds of oncogenes at single cell level simultaneously, and confirm the hypothesis of 'model of stepwise carcinogenesis'. Methods: The method of non-isotope double in situ hybridization was established with the digoxigenin (Dig) and biotin(Bio) labelled probes. The expression of two members of oncogenes of the myc and/or ras gene families (myc and N-ras, myc and K-ras, myc and H-ras, N-ras and K-ras , N-ras and H-ras, K-ras and H-ras) was further studied with the method in 10 cases of Chinese Hepatocellular Carcinomas (HCC). Results : Co-expression of two kinds of oncogenes was detected only in a few cases, reflected by coshowing two different hybridization signals ,i. e. , co-showing of Dig-myc and Bio-H-ras, Dig-myc and Bio-N-ras, Dig-myc and Bio-K-ras was observed in 2 cases, 2 cases and one case, respectively. The common characteristics were that positive cells of myc distributed diffusely, while positive cells of ras diatributed sporadically or locally among the positive cells of myc, and only a few cells exhibited co-showing of two oncogenes at single cell level. There were only 2 cases representing co-showing of two oncogenes in ras gene family (Dig-N-ras and bio-H-ras, Dig-N-ras and Bio-K-ras , respectively). The two kinds of positive cells of different ras gene represented mixed local and sporadical distribution, and co-showing of two signals was found in a few cells at single cell level. Conclusion: There are multiple oncogenes involving in tumorigenesis by their ordered activation. The activation of the ras gene family plays a role in promotion, while the activation of myc is an important event in late stage of tumorigenesis展开更多
Rxol cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with ...Rxol cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with Hpa I and Xma I. pMNDRBBin6 carrying the gene Rxo1 was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxo1 gene had been cloned into pMNDRBBin6. This double right-border binary vector, named as pMNDRBBin6-Rxol, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation.展开更多
Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB）...Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.展开更多
In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrho...In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrhosis.Among the most prevalent defects in a wide range of tumours,notably HCC,is the silencing of the p53 tumour suppressor gene.The control of the cell cycle and the preservation of gene function are both critically important functions of p53.In order to pinpoint the core mechanisms of HCC and find more efficient treatments,molecular research employing HCC tissues has been the main focus.Stimulated p53 triggers necessary reactions that achieve cell cycle arrest,genetic stability,DNA repair and the elimination of DNA-damaged cells’responses to biological stressors(like oncogenes or DNA damage).To the contrary hand,the oncogene protein of the murine double minute 2(MDM2)is a significant biological inhibitor of p53.MDM2 causes p53 protein degradation,which in turn adversely controls p53 function.Despite carrying wt-p53,the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway.High p53 in-vivo expression might have two clinical impacts on HCC:(1)Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways;and(2)Exogenous p53 makes HCC susceptible to various anticancer drugs.This review describes the functions and primary mechanisms of p53 in pathological mechanism,chemoresistance and therapeutic mechanisms of HCC.展开更多
[Objectives]The CRISPR/Cas9(Clustered regulatory interspaced short palindromic repeat/Cas9)gene editing technology is the third generation of"genome fixed-point editing technology"following the"zinc fin...[Objectives]The CRISPR/Cas9(Clustered regulatory interspaced short palindromic repeat/Cas9)gene editing technology is the third generation of"genome fixed-point editing technology"following the"zinc finger endonuclease(ZFN)"and"transcription activator effector nuclease(TALEN)".Glucotransferase genes UGT84A2 and UGT84A4,can simultaneously convert hydroxycinnamate into 1-O-β-glucose esters as isozymes.The CRISPR/Cas9 technology was used to construct double mutants of Arabidopsis thaliana ugt84a2/ugt84a4.[Methods]A CRISPR/Cas9 double mutant expression vector was constructed using UGT84A2 and UGT84A4 as the target genes.The Agrobacterium-mediated dip dyeing method was used to transform wild-type A.thaliana,and the CRISPR/Cas9system was used to target and knock out A.thaliana UGT84A2 and UGT84A4 genes.[Results]The descendants of A.thaliana with the UGT84A2/UGT84A4 gene were sequenced and analyzed.Thirteen positively transformed plants obtained were analyzed according to the sequencing results,and the ugt84a2/ugt84a4 double mutants were screened.[Conclusions]This study provides a reference for the functional study of UGT84A2 and UGT84A4 isoenzyme genes in other species,as well as strong theoretical and method support for accelerating the development and utilization of UGT84A2/UGT84A4 functional gene resources.展开更多
In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.H...In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.Human G-CSF cDNA was inserted into the Bgl Ⅱ site of the polyclonal sites within the U3 region of the 3'long terminal repeat(3'-LTR) in the vector(N2A). After being identified, the gene was transduced into Ψ-2 packaging cell line by using the electroporation method. Consequently, the gene was duplicated in the infected cells, and transferred to the 5'-LRT, and then placed outside the retroviral transcriptional unit. After two weeks the neomycin resistance positive colonies were grown in the G-418 medium. The supernatant virus was titred and then transferred to NIH3T3 cells. The G-CSF value of positive clone N2AG7-4 was up to 53. 3 U/106 cells. Southern blot and Northern blot analysis showed that the chimeric gene faithfully duplicated in the cells infected with the corresponding virus and generated two copies with one in each LTR.展开更多
目的比较伴或不伴基因突变的原发性肺腺癌在体积倍增时间(volume doubling time,VDT)和质量倍增时间(mass doubling time,MDT)间的差异。方法选取2019年1月—2020年12月在同济大学附属上海市肺科医院进行手术治疗且术前至少进行2次胸部...目的比较伴或不伴基因突变的原发性肺腺癌在体积倍增时间(volume doubling time,VDT)和质量倍增时间(mass doubling time,MDT)间的差异。方法选取2019年1月—2020年12月在同济大学附属上海市肺科医院进行手术治疗且术前至少进行2次胸部非增强CT扫描的患者为研究对象。根据放射科医师手工分割的三维掩模计算VDT和MDT。采用Bland-Altman方法进行观察者内变异性评估。采用Mann-Whitney U检验比较驱动基因有无突变肿瘤的VDT和MDT差异。采用Kruskal-Wallis检验比较不同EGFR突变位点VDT和MDT的差异。结果共计279例患者(男性99例,女性280例),平均年龄(62.15±8.90)岁,共287个结节。根据驱动基因状态分为突变组72例,野生组215例,基因突变发生率为74.9%(215/287)。突变组和野生组MDT在驱动基因状态上的差异有统计学意义(537 d vs 824 d,P=0.004),VDT的差异无统计学意义(767 d vs 593 d)。EGFR阳性腺癌的MDT比EGFR阴性腺癌长,但VDT差异并不显著(VDT,758 d vs 593 d,P=0.382;MDT,824 d vs 537 d,P=0.004)。在生长结节中,驱动基因阳性腺癌的VDT和MDT均比野生型腺癌长(VDT,759 d vs 592 d,P=0.048;MDT,749 d vs 499 d,P<0.001;VDT,768 d vs 593 d,P=0.081,MDT,737 d vs 518 d,P=0.001)。结论在原发性浸润性肺腺癌中,驱动基因阳性(尤其是EGFR阳性)的肿瘤倍增时间更长,且在生长中的结节中更为显著。展开更多
A DH population derived from C49S-87/01Y1-1069 was used to study the inheritance of wheat haploid embryo production frequency（EPF） in wheat × maize cross with the mixed major gene and polygene inheritance model...A DH population derived from C49S-87/01Y1-1069 was used to study the inheritance of wheat haploid embryo production frequency（EPF） in wheat × maize cross with the mixed major gene and polygene inheritance model of quantitative traits. The results showed that the EPF of wheat × maize cross was controlled by two dominant epistatic genes and polygene with gene effects of 1.95 for the first major gene, 6.69 for the second one and 2.80 for the polygene. The inheritability of major genes was as high as 72.09%, suggesting that the differences in EPF among wheat materials were mainly influenced by genotype. However, non-genetic factors were still important, especially for wheat materials with low EPF.展开更多
Rice with low glutelin content is suitable as functional food for patients affected by kidney failure. Low glutelincontent gene Lgc1 in rice has a 3.5-kb deletion between two highly similar glutelin genes GluB4 and Gl...Rice with low glutelin content is suitable as functional food for patients affected by kidney failure. Low glutelincontent gene Lgc1 in rice has a 3.5-kb deletion between two highly similar glutelin genes GluB4 and GluB5, which locates on the short arm of chromosome 2. To improve the selection efficiency in low glutelin-content rice breeding, two molecular markers designated as InDel-Lgc1-1 and InDel-Lgc1-2 were developed to detect the low glutelin-content gene Lgc1. A double PCR detection indicated that combined use of the two markers could easily distinguish the genotypes of Lgc1 from different rice varieties. Therefore, as a simple and low-cost technique, the molecular marker could be widely used to identify different varieties with Lgc1 gene and applied in marker-assisted selection of low glutelin-content rice.展开更多
AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an...AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an adenoviral gene vector. METHODS： Human KDR promoter was cloned by polymerase chain reaction （PCR）, and two recombinant adenoviral plasmids pAdKDR-CdgIyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses （AdKDR-CDglyTK and AdCMV-CDglyTK） respectively, and the infection rates were estimated by detection of green fluorescent protein （GFP） expression. Infected cells were cultured in culture media containing different concentrations of 5-fiuoroo/tosine （5-FC） and ganciclovir （GCV）, and the killing effects were measured. RESULTS： The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs： HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVo CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK （P 〈 0.001）. CONCLUSION： Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.展开更多
Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacill...Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.展开更多
This study was conducted to assess the occurrence and nature of extended-spectrum beta lactamase (ESBL) producing <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em> isolates from pati...This study was conducted to assess the occurrence and nature of extended-spectrum beta lactamase (ESBL) producing <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em> isolates from patients who presented with urinary tract infection at Federal Teaching Hospital Gombe. Isolates collected were recovered on MacConkey agar at 35<span style="white-space:nowrap;">°</span>C and were identified as members of Enterobacteriaceae, and further screened for antimicrobial susceptibility and resistance by disc diffusion method. Isolates resistant to oxyimino-cephalosporins were confirmed as ESBL producers using Double Disks Synergy Test (DDST). The study shows 66% resistance to ceftriaxone (30 μg) in <em>K. pneumoniae</em>, which was the highest value recorded and a 51% resistance to cefpodoxime (10 <em>μ</em>g) in <em>E. coli</em>. The sensitivity of <em>E. coli </em>and <em>K. pneumoniae</em> isolates to cefpodoxime (10 <em>μ</em>g) were 49% and 33.9% respectively. ESBLs were detected among 40% (40/100) of <em>E. coli</em> and 54.13% (59/109) of <em>K. pneumoniae</em> isolates. Molecular characterization of ESBL encoding genes among <em>E. coli</em> isolates using multiplex-PCR showed 10% prevalence of SHV gene and 5% prevalence for CTX-M gene while TEM gene was not detected. In <em>K. pneumoniae</em> isolates, 5% prevalence was recorded for each of the three genes screened. The study revealed a co-occurrence of SHV and CTX-M in 75% of the <em>E. coli</em> and 70% of the <em>K. pneumoniae</em> isolates;the occurrence of all the three genes was seen in 10% and 5% of <em>K. pneumoniae</em> and <em>E. coli</em> respectively. Multiplex-PCR method provided an efficient and rapid detection of ESBL related genes, hence could be used in epidemiological studies among ESBL isolates. Monitoring dissemination and transmissions of ESBL producers are highly recommended for optimum patient care and preventing the spread of multidrug resistant (MDR) pathogens.展开更多
Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading...Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.展开更多
文摘Objective: To establish a method of non-isotope double in situ hybridization in order to detect the expression of two kinds of oncogenes at single cell level simultaneously, and confirm the hypothesis of 'model of stepwise carcinogenesis'. Methods: The method of non-isotope double in situ hybridization was established with the digoxigenin (Dig) and biotin(Bio) labelled probes. The expression of two members of oncogenes of the myc and/or ras gene families (myc and N-ras, myc and K-ras, myc and H-ras, N-ras and K-ras , N-ras and H-ras, K-ras and H-ras) was further studied with the method in 10 cases of Chinese Hepatocellular Carcinomas (HCC). Results : Co-expression of two kinds of oncogenes was detected only in a few cases, reflected by coshowing two different hybridization signals ,i. e. , co-showing of Dig-myc and Bio-H-ras, Dig-myc and Bio-N-ras, Dig-myc and Bio-K-ras was observed in 2 cases, 2 cases and one case, respectively. The common characteristics were that positive cells of myc distributed diffusely, while positive cells of ras diatributed sporadically or locally among the positive cells of myc, and only a few cells exhibited co-showing of two oncogenes at single cell level. There were only 2 cases representing co-showing of two oncogenes in ras gene family (Dig-N-ras and bio-H-ras, Dig-N-ras and Bio-K-ras , respectively). The two kinds of positive cells of different ras gene represented mixed local and sporadical distribution, and co-showing of two signals was found in a few cells at single cell level. Conclusion: There are multiple oncogenes involving in tumorigenesis by their ordered activation. The activation of the ras gene family plays a role in promotion, while the activation of myc is an important event in late stage of tumorigenesis
文摘Rxol cloned from maize is a non-host gene resistant to bacterial leaf streak of rice. pCAMBIA1305-1 with Rxo1 was digested with Sca I and NgoM IV and the double right-border binary vector pMNDRBBin6 was digested with Hpa I and Xma I. pMNDRBBin6 carrying the gene Rxo1 was acquired by ligation of blunt-end and cohesive end. The results of PCR, restriction enzyme analysis and sequencing indicated that the Rxo1 gene had been cloned into pMNDRBBin6. This double right-border binary vector, named as pMNDRBBin6-Rxol, will play a role in breeding marker-free plants resistant to bacterial leaf streak of rice by genetic transformation.
基金Supported by Shandong Swine Industry Technology System and Science and Technology Planning Program for Basic Research in Qingdao City(12-1-4-14-jch)
文摘Gene targeting technology is an important means to investigate gene functions, but its efficiency of gene targeting is very low, especially for somatic cell targeting. Artificially induced double-strand breaks （DSB） and triplex forming oligonucleotide （TFO） are currently developed methods to improve the targeting efficiency. This paper summarized the basic principles, design ideas and application in gene targeting efficiency improvement of these two methods, analyzed and com- pared their characteristics, and finally proposed prospects for their future development.
文摘In the world,hepatocellular carcinoma(HCC)is among the top 10 most prevalent malignancies.HCC formation has indeed been linked to numerous etiological factors,including alcohol usage,hepatitis viruses and liver cirrhosis.Among the most prevalent defects in a wide range of tumours,notably HCC,is the silencing of the p53 tumour suppressor gene.The control of the cell cycle and the preservation of gene function are both critically important functions of p53.In order to pinpoint the core mechanisms of HCC and find more efficient treatments,molecular research employing HCC tissues has been the main focus.Stimulated p53 triggers necessary reactions that achieve cell cycle arrest,genetic stability,DNA repair and the elimination of DNA-damaged cells’responses to biological stressors(like oncogenes or DNA damage).To the contrary hand,the oncogene protein of the murine double minute 2(MDM2)is a significant biological inhibitor of p53.MDM2 causes p53 protein degradation,which in turn adversely controls p53 function.Despite carrying wt-p53,the majority of HCCs show abnormalities in the p53-expressed apoptotic pathway.High p53 in-vivo expression might have two clinical impacts on HCC:(1)Increased levels of exogenous p53 protein cause tumour cells to undergo apoptosis by preventing cell growth through a number of biological pathways;and(2)Exogenous p53 makes HCC susceptible to various anticancer drugs.This review describes the functions and primary mechanisms of p53 in pathological mechanism,chemoresistance and therapeutic mechanisms of HCC.
基金Supported by Natural Science Foundation of Shandong Province(ZR2017PC007)Project of Shandong(Linyi)Institute of Modern Agriculture of Zhejiang University for Serving Local Economic Development(ZDNY-2020-FWLY02007)Doctoral Program of China West Normal University(18Q051)。
文摘[Objectives]The CRISPR/Cas9(Clustered regulatory interspaced short palindromic repeat/Cas9)gene editing technology is the third generation of"genome fixed-point editing technology"following the"zinc finger endonuclease(ZFN)"and"transcription activator effector nuclease(TALEN)".Glucotransferase genes UGT84A2 and UGT84A4,can simultaneously convert hydroxycinnamate into 1-O-β-glucose esters as isozymes.The CRISPR/Cas9 technology was used to construct double mutants of Arabidopsis thaliana ugt84a2/ugt84a4.[Methods]A CRISPR/Cas9 double mutant expression vector was constructed using UGT84A2 and UGT84A4 as the target genes.The Agrobacterium-mediated dip dyeing method was used to transform wild-type A.thaliana,and the CRISPR/Cas9system was used to target and knock out A.thaliana UGT84A2 and UGT84A4 genes.[Results]The descendants of A.thaliana with the UGT84A2/UGT84A4 gene were sequenced and analyzed.Thirteen positively transformed plants obtained were analyzed according to the sequencing results,and the ugt84a2/ugt84a4 double mutants were screened.[Conclusions]This study provides a reference for the functional study of UGT84A2 and UGT84A4 isoenzyme genes in other species,as well as strong theoretical and method support for accelerating the development and utilization of UGT84A2/UGT84A4 functional gene resources.
文摘In order to overcome the interference of the internal promoter in retroviral vector with a foreign DNA expression, we used the double-copy vector(DC vector) system to improve the effect of transduced gene expression.Human G-CSF cDNA was inserted into the Bgl Ⅱ site of the polyclonal sites within the U3 region of the 3'long terminal repeat(3'-LTR) in the vector(N2A). After being identified, the gene was transduced into Ψ-2 packaging cell line by using the electroporation method. Consequently, the gene was duplicated in the infected cells, and transferred to the 5'-LRT, and then placed outside the retroviral transcriptional unit. After two weeks the neomycin resistance positive colonies were grown in the G-418 medium. The supernatant virus was titred and then transferred to NIH3T3 cells. The G-CSF value of positive clone N2AG7-4 was up to 53. 3 U/106 cells. Southern blot and Northern blot analysis showed that the chimeric gene faithfully duplicated in the cells infected with the corresponding virus and generated two copies with one in each LTR.
文摘目的比较伴或不伴基因突变的原发性肺腺癌在体积倍增时间(volume doubling time,VDT)和质量倍增时间(mass doubling time,MDT)间的差异。方法选取2019年1月—2020年12月在同济大学附属上海市肺科医院进行手术治疗且术前至少进行2次胸部非增强CT扫描的患者为研究对象。根据放射科医师手工分割的三维掩模计算VDT和MDT。采用Bland-Altman方法进行观察者内变异性评估。采用Mann-Whitney U检验比较驱动基因有无突变肿瘤的VDT和MDT差异。采用Kruskal-Wallis检验比较不同EGFR突变位点VDT和MDT的差异。结果共计279例患者(男性99例,女性280例),平均年龄(62.15±8.90)岁,共287个结节。根据驱动基因状态分为突变组72例,野生组215例,基因突变发生率为74.9%(215/287)。突变组和野生组MDT在驱动基因状态上的差异有统计学意义(537 d vs 824 d,P=0.004),VDT的差异无统计学意义(767 d vs 593 d)。EGFR阳性腺癌的MDT比EGFR阴性腺癌长,但VDT差异并不显著(VDT,758 d vs 593 d,P=0.382;MDT,824 d vs 537 d,P=0.004)。在生长结节中,驱动基因阳性腺癌的VDT和MDT均比野生型腺癌长(VDT,759 d vs 592 d,P=0.048;MDT,749 d vs 499 d,P<0.001;VDT,768 d vs 593 d,P=0.081,MDT,737 d vs 518 d,P=0.001)。结论在原发性浸润性肺腺癌中,驱动基因阳性(尤其是EGFR阳性)的肿瘤倍增时间更长,且在生长中的结节中更为显著。
基金Supported by National High Technology Research and Development Program of China(863 Program)(2011AA10A106)Yunnan Provincial Fund for Applied Basic Researches(2010CC001)Key New Product Development Plan of Yunnan Province(2012BB015)~~
文摘A DH population derived from C49S-87/01Y1-1069 was used to study the inheritance of wheat haploid embryo production frequency（EPF） in wheat × maize cross with the mixed major gene and polygene inheritance model of quantitative traits. The results showed that the EPF of wheat × maize cross was controlled by two dominant epistatic genes and polygene with gene effects of 1.95 for the first major gene, 6.69 for the second one and 2.80 for the polygene. The inheritability of major genes was as high as 72.09%, suggesting that the differences in EPF among wheat materials were mainly influenced by genotype. However, non-genetic factors were still important, especially for wheat materials with low EPF.
基金supported by the National Transgenic Crops Program, China (Grant No. 2008ZX08001-006)the Research Funds for Public Benefit in Ministry of Agriculture, China (Grant No. 200803056)+1 种基金the Key Support Program of Jiangsu Science and Technology, China (Grant No. BE2008354)the Self-directed Innovation Fund of Agricultural Science and Technology in Jiangsu Province, China (Grant No. CX [08]603)
文摘Rice with low glutelin content is suitable as functional food for patients affected by kidney failure. Low glutelincontent gene Lgc1 in rice has a 3.5-kb deletion between two highly similar glutelin genes GluB4 and GluB5, which locates on the short arm of chromosome 2. To improve the selection efficiency in low glutelin-content rice breeding, two molecular markers designated as InDel-Lgc1-1 and InDel-Lgc1-2 were developed to detect the low glutelin-content gene Lgc1. A double PCR detection indicated that combined use of the two markers could easily distinguish the genotypes of Lgc1 from different rice varieties. Therefore, as a simple and low-cost technique, the molecular marker could be widely used to identify different varieties with Lgc1 gene and applied in marker-assisted selection of low glutelin-content rice.
基金Supported by the Natural Science Foundation of Guangdong Province,No.013072the 863 Program Funds,No.2001AA 217171
文摘AIM： To study the selective killing of human umbilical vein endothelial cells （HUVECs） by a double suicide gene under the regulation of a kinase domain insert containing receptor （KDR） promoter and mediated by an adenoviral gene vector. METHODS： Human KDR promoter was cloned by polymerase chain reaction （PCR）, and two recombinant adenoviral plasmids pAdKDR-CdgIyTK, pAdCMV-CDglyTK were constructed according to a two-step transformation protocol. These two newly constructed plasmids were then transfected into 293 packaging cells to grow adenovirus, which were further multiplied and purified. HUVECs and LoVo cells were infected with either of the two resultant recombinant adenoviruses （AdKDR-CDglyTK and AdCMV-CDglyTK） respectively, and the infection rates were estimated by detection of green fluorescent protein （GFP） expression. Infected cells were cultured in culture media containing different concentrations of 5-fiuoroo/tosine （5-FC） and ganciclovir （GCV）, and the killing effects were measured. RESULTS： The two recombinant adenoviral plasmids pAdKDR-CdglyTK, pAdCMV-CDglyTK were successfully constructed and transfected into 293 cells. The resultant recombinant adenoviruses infected cells caused similar infection rates; and the infected cells exhibited different sensitivity to the prodrugs： HUVECs infected with AdCMV-CDglyTK and LoVo cells infected with AdCMVo CDglyTK were highly sensitive to the prodrugs, and HUVECs infected with AdKDR-CDglyTK were similarly sensitive but significantly more sensitive than the LoVo cells infected with AdKDR-CdglyTK （P 〈 0.001）. CONCLUSION： Selective killing of HUVECs may be achieved by gene transfer of double suicide gene under the regulation of the KDR promoter. This finding may provide an optional way to target gene therapy of malignant tumors by abrogation of tumor blood vessels.
基金Higher Education Commission,Pakistan for providing funds。
文摘Transgenic cotton carrying the CrylAc gene has revolutionized insect pest control since its adoption,although the development of resistance in insect pests has reduced its efficacy.After 10 years of cultivating Bacillus thuringiensis(Bt)cotton with a single Cry1 Ac gene,growers are on the verge of adopting Bt cotton that carries the double gene(Cry1 Ac+Cry2 A)due to its better effectiveness against insect pests.Thus,the current study was designed to evaluate the role of each gene in the effectiveness of Bt cotton carrying the double gene.The expression levels of the Cry1 Ac and Cry2 A genes were evaluated in the leaves of 10 genotypes(2 parents and 8 Fhybrids)at 30 days after sowing(DAS),while samples of leaves,bolls and flowers were taken from the upper and lower canopies at 70 and 110 DAS.The Fhybrids were developed through reciprocal crosses between two Bt(CKC-1,CKC-2)and two non-Bt(MNH-786,FH-942)parents.The differential expression of transgenes was evaluated through Enzyme Linked Immuno-Sorbent Assay(ELISA).The results showed that the MNH786 xCKC-1 hybrid had the highest concentrations of Cry1 Ac gene at30 DAS(3.08μg g^(-1))and 110 DAS(1.01μg g^(-1))in leaves.In contrast,the CKC-2 xMNH-786 hybrid showed the lowest concentrations of Cry1 Ac gene at 30 DAS(2.30μg g^(-1))and 110 DAS(0.86μg g^(-1)).The Fhybrid FH-942×CKC-2 showed the highest concentrations of Cry2 A gene at 30 DAS(8.39μg g^(-1))and 110 DAS(7.74μg g^(-1))in leaves,while the CKC-1 xMNH-786 hybrid expressed the lowest concentrations of Cry2 A gene at 30 DAS(7.10μg g^(-1))and 110 DAS(8.31μg g^(-1)).A comparison between the two stages of plant growth showed that leaves had the highest concentrations at 30 DAS,whereas the lowest concentrations were observed at 110 DAS for both genes in leaves.When the expression pattern was compared between various plant parts in genotype CKC-2,it was found that leaves had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))at 70 DAS,followed by bolls(Cry1 Ac(1.66μg g^(-1))and Cry2 A(8.15μg g^(-1)))and flowers(Cry1 Ac(1.07μg g^(-1))and Cry2 A(7.99μg g^(-1))).The genotype CKC-2 had higher concentrations of Cry1 Ac(3.12μg g^(-1))and Cry2 A(8.31μg g^(-1))in the upper canopy but less accumulation(2.66μg g^(-1)of Cry1 Ac,8.09μg g^(-1)of Cry2 A)in the lower canopy at 70 DAS.Similarly,at 110 DAS,the expression levels of Cry1 Ac and Cry2 A in upper and lower canopy leaves were 1.52 and 7.92μg 9,and 0.99 and 7.54μg 9,respectively.Hence,the current study demonstrates that different genotypes showed variable expression for both of the Cry1 Ac and Cry2 A genes during plant growth due to different genetic backgrounds.The Cry2 A gene had three-fold higher expression than Cry1 Ac with significant differences in expression in different plant parts.The findings of this study will be helpful for breeding insect-resistant double-gene genotypes with better gene expression levels of Cry1 Ac and Cry2 A for sustainable cotton production worldwide.
文摘This study was conducted to assess the occurrence and nature of extended-spectrum beta lactamase (ESBL) producing <em>Escherichia coli</em> and <em>Klebsiella pneumoniae</em> isolates from patients who presented with urinary tract infection at Federal Teaching Hospital Gombe. Isolates collected were recovered on MacConkey agar at 35<span style="white-space:nowrap;">°</span>C and were identified as members of Enterobacteriaceae, and further screened for antimicrobial susceptibility and resistance by disc diffusion method. Isolates resistant to oxyimino-cephalosporins were confirmed as ESBL producers using Double Disks Synergy Test (DDST). The study shows 66% resistance to ceftriaxone (30 μg) in <em>K. pneumoniae</em>, which was the highest value recorded and a 51% resistance to cefpodoxime (10 <em>μ</em>g) in <em>E. coli</em>. The sensitivity of <em>E. coli </em>and <em>K. pneumoniae</em> isolates to cefpodoxime (10 <em>μ</em>g) were 49% and 33.9% respectively. ESBLs were detected among 40% (40/100) of <em>E. coli</em> and 54.13% (59/109) of <em>K. pneumoniae</em> isolates. Molecular characterization of ESBL encoding genes among <em>E. coli</em> isolates using multiplex-PCR showed 10% prevalence of SHV gene and 5% prevalence for CTX-M gene while TEM gene was not detected. In <em>K. pneumoniae</em> isolates, 5% prevalence was recorded for each of the three genes screened. The study revealed a co-occurrence of SHV and CTX-M in 75% of the <em>E. coli</em> and 70% of the <em>K. pneumoniae</em> isolates;the occurrence of all the three genes was seen in 10% and 5% of <em>K. pneumoniae</em> and <em>E. coli</em> respectively. Multiplex-PCR method provided an efficient and rapid detection of ESBL related genes, hence could be used in epidemiological studies among ESBL isolates. Monitoring dissemination and transmissions of ESBL producers are highly recommended for optimum patient care and preventing the spread of multidrug resistant (MDR) pathogens.
文摘Gene editing has recently emerged as a promising technology to engineer genetic modifications precisely in the genome to achieve long-term relief from corneal disorders.Recent advances in the molecular biology leading to the development of clustered regularly interspaced short palindromic repeats(CRISPRs) and CRISPR-associated systems,zinc finger nucleases and transcription activator like effector nucleases have ushered in a new era for high throughput in vitro and in vivo genome engineering.Genome editing can be successfully used to decipher complex molecular mechanisms underlying disease pathophysiology,develop innovative next generation gene therapy,stem cell-based regenerative therapy,and personalized medicine for corneal and other ocular diseases.In this review we describe latest developments in the field of genome editing,current challenges,and future prospects for the development of personalized genebased medicine for corneal diseases.The gene editing approach is expected to revolutionize current diagnostic and treatment practices for curing blindness.
