50 results about "Intervening Sequence" patented technology

A gene activation / inactivation and site-specific integration system has been developed for mammalian cells. The invention system is based on the recombination of transfected sequences by FLP, a recombinase derived from Saccharomyces. In several cell lines, FLP has been shown to rapidly and precisely recombine copies of its specific target sequence. For example, a chromosomally integrated, silent β-galactosidase reporter gene was activated for expression by FLP-mediated removal of intervening sequences to generate clones of marked cells. Alternatively, the reverse reaction can be used to target transfected DNA to specific chromosomal sites. These results demonstrate that FLP can be used, for example, to mosaically activate or inactivate transgenes for a variety of therapeutic purposes, as well as for analysis of vertebrate development. The FLP recombination system of the present invention can be incorporated in transgenic, non-human mammals to achieve site-specific integration of transgenes, to construct functional genes or to disrupt existing genes.

A recombinant nucleic acid used for the production of a defective adenovirus containing an inserted sequence coding for a cytokine under the control of a promoter in the genomic sequence of the recombinant adenovirus. This recombinant adenovirus is useful in the preparation of anti-tumoral drugs which can be directly injected into the tumor of the host.

The present invention relates to a process for synthesizing or amplifying efficiently a nucleic acid comprising a target nucleic acid sequence. The process involves providing a primer comprising in its 3′-end portion a sequence (Ac′) which hybridizes a sequence (A) in the 3′-end portion of the target nucleic acid sequence, and in the 5′-side of the sequence (Ac′) a sequence (B′) which hybridizes the complementary sequence (Bc) of a sequence (B) positioned in the 5′-side of the sequence (A) on the target nucleic acid sequence, wherein {X−(Y−Y′)} / X is in the range of −1.00 to 1.00, in which X denotes the number of bases in the sequence (Ac′), Y denotes the number of bases in the region flanked by the sequences (A) and (B) in the target nucleic acid sequence, and Y′ denotes the number of bases in an intervening sequence between the sequences (Ac′) and (B′) (Y′ may be zero).

The present invention relates to a vector for the expression of recombinant proteins, antigens, pathogen-like particles and immunogenic complexes, said vector (pMRKA vector) being produced by modifying the plasmids containing the gene sequence of the T7 promoter of E. coli, this modification being mainly characterized by the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene, and by the insertion of the par sequence (partition sequence which determines the efficient segregation of the plasmids in daughter cells during cell division). Also provided are expression vectors based on the pMRKA plasmid, which additionally comprise at least one of the gene sequences of the exosome of P. abyssi, which vectors are designated pMRKA-EXO, pMRKA-RING and pSUMAC. The invention also provides the vectors additionally comprising gene sequences with immunomodulatory or immunoregulatory activity, preferably the pMRKA-Z-Z-EXO and pMRKA-Z-Z-RING vectors. Other aspects of the invention include the method for producing said expression vectors and the use of the obtained vectors.

The invention discloses a gene mutation multi-detecting method based on a signal amplification DNA logic gate. The method comprises the following steps: performing high specificity recognition on a detecting target by invading into a signal amplification reaction by utilizing nucleic acid, and simultaneously, invading into the signal amplification reaction by utilizing a controllable DNA polymerase extension reaction bridging two-step nucleic acid to form signal amplification. The method disclosed by the invention has high detecting sensitivity and high detecting specificity to a nucleic acid target. A DNA logic gate is constructed by invading into the signal amplification reaction by adopting the controllable DNA polymerase extension reaction bridging two-step nucleic acid disclosed by the invention, a background signal is inhibited by introducing an intervening sequence, and meanwhile, a two-step signal amplification reaction exists, so that the defecting sensitivity is improved, and high signal-to-noise ratio output can be realized under the conditions of trace amount of DNA input and short reaction time. Moreover, through the method established by the invention, a plurality of gene mutation points can be detected simultaneously and results can be subjected to logical judgment, and an analyzing process of gene mutation detection in a clinically individualized medicating process is simplified.

Compositions and methods are provided for the treatment and diagnosis of diseases amenable to treatment through modulation of the synthesis or metabolism of intercellular adhesion molecules. In accordance with preferred embodiments, oligonucleotides are provided which are specifically hybridizable with nucleic acids encoding intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1. The oligonucleotide comprises nucleotide units sufficient in identity and number to effect said specific hybridization. In other preferred embodiments, the oligonucleotides are specifically hybridizable with a transcription initiation site, a translation initiation site, 5'-untranslated sequences, 3'-untranslated sequences, and intervening sequences. Methods of treating animals suffering from disease amenable to therapeutic intervention by modulating cell adhesion proteins with an oligonucleotide specifically hybridizable with RNA or DNA corresponding to one of the foregoing proteins are disclosed. Methods for treatment of diseases responding to inhibition of cell adhesion molecules are disclosed.

Gene of an E.coli-Lactobacillus shuttle plasmid expressing and secreting beta-galactosidase is composed of the gene encoding beta-galactosidase and its upstream regulatory sequence containing SD sites. Experiments proves that the recombinant plasmid can be transformed into Lactococcus lactis, and highly express the non-fused beta-galactosidase in E.coli and Lactococcus lactis, which is applicable in the lactic acid bacteria strain expressing beta-galactosidaseion. The construction method includes the following steps: taking Lactobacillus Bulgaria subspecies genomic DNA as template to amplify and obtain the beta-galactosidase gene containing the upstream regulatory sequence, digesting the gene to conjugate with the dephosphorylated vector digested with the same two enzymes and transform E.coli, then screening the positive one to extract the recombinant for enzyme identification and sequencing the inserted sequence.

A thermophilic microorganism lacks lactate dehydrogenase activity and preferably contains an active pyruvate formate lyase pathway. The thermophilic microorganism contains a gene encoding an NAD-linked formate dehydrogenase. The gene encoding an NAD-linked formate dehydrogenase is preferably a codon optimised version of the gene encoding a thermostable NAD-linked formate dehydrogenase. DNA constructs allow stable expression of the gene encoding an NAD-linked formate dehydrogenase in the thermophilic microorganism. The DNA constructs are based upon use of an insertion sequence to achieve stable expression or recombination to insert the gene encoding an NAD-linked formate dehydrogenase into the lactate dehydrogenase gene, thus achieving gene knockout and new functionality in a single step. The microorganisms are useful in fermentation of sugars to produce ethanol.

The invention discloses a method for verifying the feasibility of inserting a CRISPR-Cas9 system mediated target gene into candida utilis. The method includes the following steps that S1, an expression vector of the candida utilis containing Cas9 segments is constructed; S2, tRNAGlu genes and downstream expression units of sgRNA are amplified, PCR link is overlapped and a restriction enzyme cutting site is introduced, and after connection, the amplified tRNAGlu genes and downstream expression units of the sgRNA are combined with the vector of the candida utilis to obtain sgRNA initial recombinant plasmid; S3, pre-insertion and target sequences are selected, a sticky tail end is added to the 5' end of a target and complementary sequence, and double chain segments are formed by annealing andthen connected with the initial recombinant plasmid after enzyme digestion to obtain sgRNA mature recombinant plasmid; S4, a recombinant donor segment containing a left homologous arm and a right homologous arm of a pre-insertion sequence is constructed; and S5, the sgRNA recombinant plasmid and the recombinant donor segment are used for transforming the candida utilis carrying Cas9. The feasibility of inserting the CRISPR-Cas9 system mediated target gene into the candida utilis is verified by identifying the expression of the donor segment.

Methods are provided for generating highly diverse libraries of expression vectors encoding fusion proteins such as single-chain antibodies via homologous recombination in yeast. The method comprises: transforming into yeast cells a linearized yeast expression vector having a 5'- and 3'-terminus sequence at the site of linearization and a library of insert nucleotide sequences that are linear and double-stranded; and having homologous recombination occur between the vector and the insert sequence such that the insert sequence is included in the vector in the transformed yeast cells. The insert sequence comprises a first nucleotide sequence encoding a first polypeptide subunit, a second nucleotide sequence encoding a second polypeptide subunit, a linker sequence encoding a linker peptide that links the first and second polypeptide subunits, and a 5'- and 3'-flanking sequence at the ends of the insert sequence which are sufficiently homologous to the 5'- and 3'-terminus sequences of the linearized yeast expression vector, respectively, to enable homologous recombination to occur. The first polypeptide subunit, the second polypeptide subunit, and the linker polypeptide are expressed as a single fusion protein; and the first and second nucleotide sequences each independently varies within the library of expression vectors.

The present invention provides an advance in phage display technology by permitting the uncoupling of the propagation of phages containing inserted sequences encoding heterologous polypeptides from the expression of said polypeptides. The invention provides phage constructs and methods for their use to permit phage coat protein expression, and thus phage propagation, in the absence of display of heterologous polypeptides, which may be expressed as a fusion with said coat protein in a regulated manner.

The invention relates to genetic engineering, in particular to a method for transferring exogenous genes into spiral seaweeds with transposons as a medium. Multiple sets of primers IS1-IS4 are designed according to sequences of known blue-green algae of the NCBI, and transposons elements are cloned from the blue-green algae and implanted into the sequences; ultrasonic transformation and screening of donor plasmids pEGFP-IS-IS-Km are conducted; EcoR I is utilized for digesting pEGFP-IS-IS-Km to obtain linear plasmids, the two ends each have an IS sequence, spiral seaweeds 869s are transformed, and GFP and G418 (npt II) are utilized for screening out mutation-inserted spiral seaweed plants. An integrated spiral seaweed screening system including gfp genes, npt II mark genes and transposons sequences is built, and the gene structure of spiral seaweeds is influenced by random insertion mutation of transposons, so that the quality of spiral seaweeds is changed. The method can be applied to building of a spiral seaweed mutation library, and can also realize introduction and expression of exogenous genes and antisense inhibition regulation of endogenous genes of spiral seaweeds.

Compositions and methods are provided for the treatment and diagnosis of diseases amenable to treatment through modulation of the synthesis or metabolism of intercellular adhesion molecules. In accordance with preferred embodiments, oligonucleotides are provided which are specifically hybridizable with nucleic acids encoding intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and endothelial leukocyte adhesion molecule-1. The oligonucleotide comprises nucleotide units sufficient in identity and number to effect said specific hybridization. In other preferred embodiments, the oligonucleotides are specifically hybridizable with a transcription initiation site, a translation initiation site, 5'-untranslated sequences, 3'-untranslated sequences, and intervening sequences. Methods of treating animals suffering from disease amenable to therapeutic intervention by modulating cell adhesion proteins with an oligonucleotide specifically hybridizable with RNA or DNA corresponding to one of the foregoing proteins are disclosed. Methods for treatment of diseases responding to modulation cell adhesion molecules are disclosed.