[Objective]The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat ...[Objective]The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%,50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture,neo gene was as screened gene,genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium,100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%). Compared with control,con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%),confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell,inserting and diagnosing ideal vector,and can save expense and time for transgenic animal production.展开更多
RNA-seq and single-cell genomic research emerge as an important research area in the recent years due to its ability to examine genetic information of any number of single cells in all living organisms.The knowledge g...RNA-seq and single-cell genomic research emerge as an important research area in the recent years due to its ability to examine genetic information of any number of single cells in all living organisms.The knowledge gained from RNA-seq and single-cell genomic research will have a great impact in many aspects of plant biology.In this review,we summary and discuss the biological significance of RNA-seq and single-cell genomic research in plants including the single-cell DNA-sequencing,RNA-seq and single-cell RNA sequencing in woody plants,methods of RNA-seq and single-cell RNA-sequencing,single-cell RNA-sequencing for studying plant development,and single-cell RNA-sequencing for elucidating cell type composition.We will focus on RNA-seq and single-cell RNA sequencing in woody plants,understanding of plant development through single-cell RNAsequencing,and elucidation of cell type composition via single-cell RNA-sequencing.Information presented in this review will be helpful to increase our understanding of plant genomic research in a way with the power of plant single-cell RNA-sequencing analysis.展开更多
Cells are the basic unit of life structure and life activities.Because of the complex micro-environment of cells,the content of components that play a key role is relatively small,so single-cell analysis is extremely ...Cells are the basic unit of life structure and life activities.Because of the complex micro-environment of cells,the content of components that play a key role is relatively small,so single-cell analysis is extremely challenging.In recent years,single-cell sequencing technology has been developed and matured.Single-cell sequencing can reveal the composition and physiological diversity of cells,and the existing single-cell separation technology,single-cell whole genome amplification technology,single The principles and applications of cell whole transcriptome amplification technology and single cell transcriptome sequencing are summarized and summarized.展开更多
Rapidly developing sequencing technologies and bioinformatic approacheshave provided us with an unprecedented instrument allowing for an unbiased and exhaustive characterization of the cancer genome in genetic, epigen...Rapidly developing sequencing technologies and bioinformatic approacheshave provided us with an unprecedented instrument allowing for an unbiased and exhaustive characterization of the cancer genome in genetic, epigenetic and transcriptomic dimensions. This review introduces recent excitingfindings and new methodologies in genomic breast cancer research. With this development, cancer genome research will illuminate new delicate interactionsbetween molecular networks and thereby unravelthe underlying biological mechanisms for cancer initiation and progression. It also holds promise for providing a molecular clock for the estimation of the temporal processes of tumorigenesis. These methods in combination with single cell sequencing will make it possible to construct a family tree elucidating the evolutionary lineage relationships between cell populations at single-cell resolution. The anticipatedrapid progress in genomic breast cancer research should lead to anenhanced understanding of breast cancer biology andguide us towardsnovel ways to ultimatelyprevent and cure breast cancer.展开更多
Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy samp...Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,展开更多
The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) ...The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.展开更多
Blood samples have traditionally been used as the main source of DNA for genetic analysis. How-ever, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we invest...Blood samples have traditionally been used as the main source of DNA for genetic analysis. How-ever, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we investigated whether human nail clippings could be used as a source of DNA for SNP genotyping, null-allele detection, and whole-genome amplification. From extracted nail DNA, we achieved amplicons up to a length of ~400 bp and >96% concordance for SNP genotyping and 100% concordance for null-allele detection compared to DNA derived from matched blood sam-ples. For whole-genome amplification, OmniPlex performed better than Multiple Displacement Amplification with a success rate of 89.3% and 76.8% for SNP genotyping and null-allele detection, respectively. Concordance was ~98% for both methods. When combined with OmniPlex whole-genome amplification, human nail clippings could potentially be used as an alternative to whole blood as a less invasive and more convenient source of DNA for genotyping studies.展开更多
AIM: To introduce an approach for the detection of putative genetic host factors that predispose patients to develop head and neck squamous cell carcinomas(HNSCC).METHODS: HNSCC most often result from the accumulation...AIM: To introduce an approach for the detection of putative genetic host factors that predispose patients to develop head and neck squamous cell carcinomas(HNSCC).METHODS: HNSCC most often result from the accumulation of somatic gene alterations found in tumor cells. A cancer-predisposing genetic background must be expected in individuals who develop multiple cancers, starting at an unexpectedly young age or with little carcinogen exposure. Genome-wide loss of heterozygosity(LOH) profiling by single nucleotide polymorphism microarray mapping was performed in a patient with a remarkable history of multifocal HNSCC.RESULTS: Regions of genomic deletions in germline DNA were identified on several chromosomes with a remarkable size between 1.6 Mb and 8.1 Mb(mega base-pair). No LOH was detected at the genomic location of the tumor suppressor gene P53.CONCLUSION: Specific patterns of germline DNA deletions may be responsible for susceptibility to HNSCC and should be further analyzed.展开更多
基金Supported by Doctoral Start Fund of Henan University of Science and Technology
文摘[Objective]The aim was to explore technical system of making single transgenic positive cells become colony cells by amplification culture. [Method] Fetal fibroblasts and mammary gland epithelial cells of single goat fetus of pBLM-C1 which specifically expressed human lactoferrin were cloned. Single cell colony of single transfection cell was prepared with 3 concentrations of 0%,50% and 100% conditioned culture media. Transfection cell and non-transfection cell were carried out amplification culture by con-culture,neo gene was as screened gene,genome DNA of transfection cell was detected by PCR method. Chromosome karyotype analysis of single colony cell was tested. [Result] Compared with non-conditioned culture medium,100% conditioned culture medium could greatly increase survived rate of single colony cells (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%). Compared with control,con-culture of transfection cell and non-transfection cell could greatly increase rate of transfection cell single colony after amplification culture (FF: 53.33% vs. 10.00%;MGE: 33.33% vs. 6.67%),confluence time of amplification culture was significantly decreased (20-30 d). The result of PCR showed that the colony cell obtained by above method contained hLF target gene. The result of karyotype analysis showed that most cloned cell chromosomes were normal. [Conclusion] The study provides a reliable method for separating transgenic cell,inserting and diagnosing ideal vector,and can save expense and time for transgenic animal production.
文摘RNA-seq and single-cell genomic research emerge as an important research area in the recent years due to its ability to examine genetic information of any number of single cells in all living organisms.The knowledge gained from RNA-seq and single-cell genomic research will have a great impact in many aspects of plant biology.In this review,we summary and discuss the biological significance of RNA-seq and single-cell genomic research in plants including the single-cell DNA-sequencing,RNA-seq and single-cell RNA sequencing in woody plants,methods of RNA-seq and single-cell RNA-sequencing,single-cell RNA-sequencing for studying plant development,and single-cell RNA-sequencing for elucidating cell type composition.We will focus on RNA-seq and single-cell RNA sequencing in woody plants,understanding of plant development through single-cell RNAsequencing,and elucidation of cell type composition via single-cell RNA-sequencing.Information presented in this review will be helpful to increase our understanding of plant genomic research in a way with the power of plant single-cell RNA-sequencing analysis.
文摘Cells are the basic unit of life structure and life activities.Because of the complex micro-environment of cells,the content of components that play a key role is relatively small,so single-cell analysis is extremely challenging.In recent years,single-cell sequencing technology has been developed and matured.Single-cell sequencing can reveal the composition and physiological diversity of cells,and the existing single-cell separation technology,single-cell whole genome amplification technology,single The principles and applications of cell whole transcriptome amplification technology and single cell transcriptome sequencing are summarized and summarized.
文摘Rapidly developing sequencing technologies and bioinformatic approacheshave provided us with an unprecedented instrument allowing for an unbiased and exhaustive characterization of the cancer genome in genetic, epigenetic and transcriptomic dimensions. This review introduces recent excitingfindings and new methodologies in genomic breast cancer research. With this development, cancer genome research will illuminate new delicate interactionsbetween molecular networks and thereby unravelthe underlying biological mechanisms for cancer initiation and progression. It also holds promise for providing a molecular clock for the estimation of the temporal processes of tumorigenesis. These methods in combination with single cell sequencing will make it possible to construct a family tree elucidating the evolutionary lineage relationships between cell populations at single-cell resolution. The anticipatedrapid progress in genomic breast cancer research should lead to anenhanced understanding of breast cancer biology andguide us towardsnovel ways to ultimatelyprevent and cure breast cancer.
基金supported by grants awarded to Yuanqing Yao by the Key Program of the "Twelfth Five-year plan" of People’s liberation Army(No.BWS11J058)the National High Technology Research and Development Program(SS2015AA020402)
文摘Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,
基金supported by grants from National Science and Technology Major Project(2012ZX10004701)
文摘The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.
文摘Blood samples have traditionally been used as the main source of DNA for genetic analysis. How-ever, this source can be difficult in terms of collection, transportation, and long-term storage. In this study, we investigated whether human nail clippings could be used as a source of DNA for SNP genotyping, null-allele detection, and whole-genome amplification. From extracted nail DNA, we achieved amplicons up to a length of ~400 bp and >96% concordance for SNP genotyping and 100% concordance for null-allele detection compared to DNA derived from matched blood sam-ples. For whole-genome amplification, OmniPlex performed better than Multiple Displacement Amplification with a success rate of 89.3% and 76.8% for SNP genotyping and null-allele detection, respectively. Concordance was ~98% for both methods. When combined with OmniPlex whole-genome amplification, human nail clippings could potentially be used as an alternative to whole blood as a less invasive and more convenient source of DNA for genotyping studies.
文摘AIM: To introduce an approach for the detection of putative genetic host factors that predispose patients to develop head and neck squamous cell carcinomas(HNSCC).METHODS: HNSCC most often result from the accumulation of somatic gene alterations found in tumor cells. A cancer-predisposing genetic background must be expected in individuals who develop multiple cancers, starting at an unexpectedly young age or with little carcinogen exposure. Genome-wide loss of heterozygosity(LOH) profiling by single nucleotide polymorphism microarray mapping was performed in a patient with a remarkable history of multifocal HNSCC.RESULTS: Regions of genomic deletions in germline DNA were identified on several chromosomes with a remarkable size between 1.6 Mb and 8.1 Mb(mega base-pair). No LOH was detected at the genomic location of the tumor suppressor gene P53.CONCLUSION: Specific patterns of germline DNA deletions may be responsible for susceptibility to HNSCC and should be further analyzed.
