Genetic manipulation of human pluripotent stem cells (hPSCs) provides a powerful tool for modeling diseases and developing future medicine. Recently a number of independent genome-editing techniques were developed, including plasmid, bacterial artificial chromosome, adeno-associated virus vector, zinc finger nuclease, transcription activator-like effecter nuclease, and helper-dependent adenoviral vector. Gene editing has been successfully employed in different aspects of stem cell research such as gene correction, mutation knock-in, and establishment of reporter cell lines (Raya et al., 2009; Howden et al., 2011; Li et al., 2011; Liu et al., 2011b; Papapetrou et al., 2011; Sebastiano et al., 2011; Soldner et al., 2011; Zou et al., 2011a). These techniques combined with the utility of hPSCs will significantly influence the area of regenerative medicine.
Cell Line ; Chromosomes, Artificial, Bacterial ; genetics ; Deoxyribonucleases ; genetics ; Dependovirus ; genetics ; Gene Targeting ; methods ; Genetic Engineering ; methods ; Genetic Vectors ; Genome, Human ; Humans ; Mutagenesis, Insertional ; Mutation ; Plasmids ; Pluripotent Stem Cells ; cytology ; metabolism ; Zinc Fingers ; genetics

The mammary gland bioreactor has great commercial value, but the examples for high level expression of foreign gene were so few that the most examples were not suitable for commercial program. The key resolution for improving the expression level of foreign gene is the construction of expression vector for mammary gland bioreactor. Nearly, many new ideas and new methods about the construction of expression vector were presented, the article summarized them.
Animals ; Animals, Genetically Modified ; Bioreactors ; Breast ; metabolism ; Chromosomes, Artificial, Yeast ; Gene Expression ; Gene Targeting ; methods ; Genetic Vectors ; Humans

The study of human new gene's function has become an increasingly active academic field basically relying on the gene knockout (KO) mouse. The construction of targeting vector economically and efficiently has turned into the key step to acquire a KO mouse because of the low efficiency of recombination with traditional constructed targeting vector. For study of the function of new gene-Resp18, we brought in a new DNA engineering platform-Red/ET recombination to construct Resp18 targeting vector. Red/ET recombineering differs from the conventional ways of vector construction (e.g., PCR, restriction enzyme digestion and ligation) and achieves genetic modification by acquisition, insertion, fusion or replacement of the target gene through small fragments mediated homologous recombination. Now Resp18 targeting vectors of three strategies were yielded successfully through two homologous recombination processes of retrieve and neo-targeting. Red/ET recombination has the advantage of getting longer homology regions without mutation, which makes it a new and reliable alternative to the construction of a targeting vector today.
Bacteriophage lambda ; genetics ; Chromosomes, Artificial, Bacterial ; genetics ; Gene Targeting ; methods ; Genetic Engineering ; methods ; Genetic Vectors ; genetics ; Plasmids ; genetics ; Viral Proteins ; genetics

Present studies describe the successful establishment of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) BAC-TO-BAC expression system. The mini-F-lacZ-attTn7-kan fragment (Luckow et al, 1993) was inserted into SeMNPV US1 isolate (SeUS1) at polyhedrin gene locus by directly cloning. The recombinant virus containing low-copy-number mini-F replication, which named bacmid, can propagate in Escherichia coli. Because SeUS1 isolate is make up of several genotypes and one bacmid carries one SeMNPV genotype, the SeUS1 BAC library is established by all SeMNPV bacmids (SeBAC). REN analysis for 111 SeBAC shows that SeUS1 consists of the genotype with whole SeMNPV genetic information and several genotypes with various different deletions. Progeny virus can be produced in insect cell line after transfection with SeBAC10, which carries the whole SeMNPV genome. So SeBAC10 is a shuttle vector that can replicate in eukaryocyte as well as prokaryocyte. Considering the insert mutation of SeMNPV polyhedrin gene (Seph) in SeBAC10, Seph was reintroduced into the bacmid by site-specific transposon-mediated insertion at attTn7, the target site for the bacterial transposon Tn7. The derived recombinant SeBAC10 was named SeBAC10ph. After SeBAC10ph was transfected into Se301 cells (a susceptible insect cell line to SeMNPV), cytopathogenic effect was shown and polyhedra appeared, which indicate that the foreign gene (Seph) is expressed.
Animals ; Cell Line ; Chromosomes, Artificial, Bacterial ; genetics ; DNA Transposable Elements ; genetics ; Genetic Vectors ; genetics ; Models, Theoretical ; Nucleopolyhedrovirus ; genetics ; physiology ; Spodoptera ; cytology ; virology ; Viral Structural Proteins ; genetics

It is clear that the construction of large insert DNA libraries is important for map-based gene cloning, the assembly of physical maps, and simple screening for specific genomic sequences. The bacterial artificial chromosome (BAC) system is likely to be an important tool for map-based cloning of genes since BAC libraries can be constructed simply and analyzed more efficiently than yeast artificial chromosome (YAC) libraries. BACs have significantly expanded the size of fragments from eukaryotic genomes that can be cloned in Escherichia coli as plasmid molecules. To facilitate the isolation of molecular-biologically important genes in Ashbya gossypii, we constructed Ashbya chromosome-specific BAC libraries using pBeloBAC11 and pBACwich vectors with an average insert size of 100 kb, which is equivalent to 19.8X genomic coverage. pBACwich was developed to streamline map-based cloning by providing a tool to integrate large DNA fragments into specific sites in chromosomes. These chromosome-specific libraries have provided a useful tool for the further characterization of the Ashbya genome including positional cloning and genome sequencing.
Ascomycota* ; Chromosomes, Artificial, Bacterial ; Chromosomes, Artificial, Yeast ; Clone Cells ; Cloning, Organism ; DNA ; Escherichia coli ; Gene Library ; Genome ; Mass Screening ; Plasmids

OBJECTIVETo annotate the human genome 3p24-p25 478 kb complete sequence.

METHODSThe protein-coding genes in the genomic sequence were identified by using ab initio gene finding, homology-based similarity database searching and all or partial mRNA aligning with genomic sequence, and the content feature of the genomic sequence were analyzed by using EMBOSS package.

RESULTSTwo known genes SLC6A1 and SLC6A11 were identified; as well as the GC content of this genomic sequence was 47% and 3 putative CpG islands were predicted in the genomic sequence, located in 130,685-131,516 bp, 307,090-307,870 bp and 415,585-416,308 bp, respectively.

CONCLUSIONSThe methods, as mentioned above, might be used for annotating the biological information in the genomic sequence, such as gene structure, GC content, CpG island.

Based on the sequence of BAC (Bacterial Artificial Chromosome) along with the Cre/lox P system, the gene-targeting vectors to multiple loci of the repetitive internal transcribed spacers between rDNA genes in Leghorn chicken were constructed. The key material of multiple loci gene targeting in vivo would be obtained. First, the plasmid of pYLSV-TDN with TK, HRDS2, and Neo genes was constructed. The TK-HRDS2-Neo DNA fragment obtained from the plasmid of pYLSV-TDN was digested by Not I/HindIII and inserted into the upstream of the lox P site of BAC plasmid for obtaining the selective vector of BAC-TDN. The expression vector of pYLVS-GID with EGFP, hIFN genes, and HRDS1 was then obtained. The plasmid of BAC-TDN-VS-GID was obtained by cotransformation of the selective vector of BAC-TDN and the expression vector of pYLVS-GID to E. coli NS3529 through the action of Cre/lox P system. The gene-targeting vector of BAC-TDN-GID to multiple loci of the ITS region in Leghorn chicken was obtained by cleaving the sequence of pYLVS with the homing endonuclease of I -Sce I and ligating with the linker of LS. The insertion and the insert direction of DNA fragments were identified by restriction digestion or PCR and sequencing in each clone. The significance of the technique ofgene-targeting vector to multiple loci are shown as follows. First, the targeting loci were increased to 100 - 300. Second, the problems of unstable expression of inserted genes were partially solved. Third, the need for safety against toxicity integration was resolved. Fourth, the forbidden zone of gene integrating on the repetitive DNA sequences was broken through.
Animals ; Attachment Sites, Microbiological ; genetics ; Chromosomes, Artificial, Bacterial ; genetics ; Cloning, Molecular ; DNA ; genetics ; metabolism ; DNA Restriction Enzymes ; metabolism ; DNA, Ribosomal Spacer ; genetics ; Escherichia coli ; genetics ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; Integrases ; genetics ; Interferon-gamma ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; genetics ; Transformation, Genetic

BACKGROUND: Williams syndrome is characterized by supravalvular aortic stenosis, mental retardation and peculiar facial appearance. Its genetic etiology is considered to be hemizygotic deletion in Chromosome 7q11.23 which includes the elastin gene. We examined the deletion in Korean Williams syndrome patients and their parents. MATERIALS AND METHOD: Sixteen patients were selected through careful clinical examination including echocardiography and cardiac angiography. Hemizygotic deletion of elastin gene was determined in patients and 21 parents with fluorescent in situ hybridization (FISH) technique using the bacterial artificial chromosome clone 244H3 probe or commercial WSCR probe. RESULTS: FISH showed hemizygotic deletion of chromosome 7 in all sixteen patients but none of their parents showed deletion. CONCLUSION: Hemizygotic deletion of elastin gene can be determined by FISH with new probe 244H3 in clinically suspected Williams syndrome patients.
Angiography ; Aortic Stenosis, Supravalvular ; Chromosomes, Artificial, Bacterial ; Chromosomes, Human, Pair 7 ; Clone Cells ; Echocardiography ; Elastin* ; Humans ; In Situ Hybridization, Fluorescence* ; Intellectual Disability ; Parents ; Williams Syndrome*

PURPOSE: Williams syndrome is characterized by supravalvular aortic stenosis, mental retardation and peculiar facial appearance. Its genetic etiology is considered to be a hemizygotic deletion in Chromosome 7q11.23, which includes the elastin gene. We examined the hemizygotic deletion of Chromosome 7q11.23 in 12 Korean Williams syndrome patients and 8 patients with isolated supravalvular aortic stenosis and performed deletion mapping in the Williams syndrome patients. METHODS: Hemizygotic deletion was determined with fluorescence in situ hybridization(FISH) using the bacterial artificial chromosome clone 244H3, which has the genomic DNA sequence of elastin gene, as a probe. For the deletion mapping, polymorphism analysis of 10 Williams syndrome patients and their parents was done with 9 dinucleotide repeat sequence polymorphic markers(D7S499, D7S672, D7S653, ELN, D7S2472, D7S1870, D7S2518, D7S675 and D7S669). RESULTS: In the Williams syndrome patients, FISH showed deletion in all. In patients with isolated supravalvular aortic stenosis, FISH showed deletion in one, partial deletion in another and no deletion in the other six patients. Polymorphism analysis showed that alleles at three loci(ELN, D7S2472 and D7S1870) were commonly deleted in the Williams syndrome patients. Paternal alleles were deleted in six patients and maternal alleles were deleted in four. CONCLUSION: Hemizygotic deletion could be detected in Williams syndrome patients with FISH and the commonly deleted loci were ELN, D7S2472 and D7S1870. Most patients with isolated supravalvular aortic stenosis showed no deletion with FISH and the genetic defect should be much smaller than what FISH could detect.
Alleles ; Aortic Stenosis, Supravalvular* ; Base Sequence ; Chromosomes, Artificial, Bacterial ; Chromosomes, Human, Pair 7* ; Clone Cells ; Dinucleotide Repeats ; Elastin ; Fluorescence ; Genes, vif ; Humans ; Intellectual Disability ; Parents ; Williams Syndrome*

PURPOSE: Williams syndrome is characterized by supravalvular aortic stenosis, mental retardation and peculiar facial appearance. Its genetic etiology is considered to be a hemizygotic deletion in Chromosome 7q11.23, which includes the elastin gene. We examined the hemizygotic deletion of Chromosome 7q11.23 in 12 Korean Williams syndrome patients and 8 patients with isolated supravalvular aortic stenosis and performed deletion mapping in the Williams syndrome patients. METHODS: Hemizygotic deletion was determined with fluorescence in situ hybridization(FISH) using the bacterial artificial chromosome clone 244H3, which has the genomic DNA sequence of elastin gene, as a probe. For the deletion mapping, polymorphism analysis of 10 Williams syndrome patients and their parents was done with 9 dinucleotide repeat sequence polymorphic markers(D7S499, D7S672, D7S653, ELN, D7S2472, D7S1870, D7S2518, D7S675 and D7S669). RESULTS: In the Williams syndrome patients, FISH showed deletion in all. In patients with isolated supravalvular aortic stenosis, FISH showed deletion in one, partial deletion in another and no deletion in the other six patients. Polymorphism analysis showed that alleles at three loci(ELN, D7S2472 and D7S1870) were commonly deleted in the Williams syndrome patients. Paternal alleles were deleted in six patients and maternal alleles were deleted in four. CONCLUSION: Hemizygotic deletion could be detected in Williams syndrome patients with FISH and the commonly deleted loci were ELN, D7S2472 and D7S1870. Most patients with isolated supravalvular aortic stenosis showed no deletion with FISH and the genetic defect should be much smaller than what FISH could detect.
Alleles ; Aortic Stenosis, Supravalvular* ; Base Sequence ; Chromosomes, Artificial, Bacterial ; Chromosomes, Human, Pair 7* ; Clone Cells ; Dinucleotide Repeats ; Elastin ; Fluorescence ; Genes, vif ; Humans ; Intellectual Disability ; Parents ; Williams Syndrome*