38 results about "Negative selection" patented technology

The invention provides an overexpression porcine co-stimulatory 4-1BB vector and application thereof. PCR (polymerase chain reaction) amplification is performed on a left homologous arm and a right homologous arm of an intron 1 of a rosa26 gene, a 4-1BB regulatory sequence and an OCT4 specific promoter; the left homologous arm, a 4-1BB expression cassette, LoxP locus-contained Cre and Neo expression cassettes, the right homologous arm and negative selection DTA diphtheria toxin are connected in sequence to obtain a 4-1BB homologous recombinant vector p4BOCNDR; the vector and a CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) targeting vector of sgRNA (small guide ribonucleic acid) containing the intron 1 of the specific targeting porcine rosa26 gene are transferred together into a porcine fetus fibroblast; by taking a positive cell as a donor cell and an oocyte as a recipient cell, a cloned embryo is obtained through a somatic cell nuclear transfer technique; the cloned embryo is transplanted into a porcine uterus for fetation to obtain a transgenic pig integrating a 4-1BB gene at the fixed point of a first intron of the rosa26 gene and automatically deleting a marker gene.

The invention relates to an allele double knockout targeting vector system and a construction method thereof. The system of the invention consists of two complementary vectors, namely pGT-V1 and pGT-V2, wherein each vector contains two in phase LoxP elements which contain a positive selection marker gene Neo/GFP and Hyg/RFP respectively; and the outside of each vector contains a negative selection marker gene TK. Meanwhile, two 8-basic group multiple cloning sites are designed and arranged between the two LoxP elements and the negative selection marker for the insertion of the homology arm. By adopting the constructed targeting vector system of the invention, two complementary targeting vectors are cotransfected into the recipient cell; through the selection of drug and fluorescent double-selection marker, the genetic modification or knockout of the two alleles of the target gene can be realized once; and the interaction of the transfected Cre enzyme and the LoxP elements can be utilized to remove the selection marker gene integrated with the genome, the time of obtaining the homozygous knockout target cell can be shortened, the safety of the transgenic animal can be increased and a valuable technology platform is provided to develop the animal transgenic researches.

The invention discloses a bacillussubtilis CRISPR-Cas9 genome editing system. A building method of the system comprises the following steps of (1) building a plasmid pBSCas9; (2) building a plasmid pDonor; (3) building an auxiliary plasmid pHG in a multi-site gRNA expression process; and (4) forming the bacillussubtilis CRISPR-Cas9 genome editing system by the pBSCas9, the pDonor and the pHG. Thebacillussubtilis CRISPR-Cas9 genome editing system solves the problems in the existing bacillussubtilis conventional genome editing technology; no negative selection marker is introduced; and a CRISPR-Cas9 and CRISPR-Cas9n genome non-trace editing system with high efficiency and high precision is built. The single-site and multi-site different-type gene editing operation can be realized; and meanwhile, the iterative editing cycle of an editing flow process can be realized.

The invention discloses a path coverage test data generation method based on a negative selection genetic algorithm which aims to ensure that generated test data has a relatively high coverage rate and contain least redundant data, thereby improving the quality of the test data and the efficiency of software testing. The method comprises the following steps: firstly, generating an initial population of a negative selection genetic algorithm in a supervised manner according to a negative selection generation strategy. Then, according to a negative selection detection strategy, dynamically optimizing population data of a negative selection genetic algorithm, and evolving to generate test data covering the target path. The test data generated by the conventional method only guarantees the coverage of a target path. Compared with the prior art, the method has the advantages that the number of generated test data cannot be guaranteed to be minimum, a large number of redundant test data arecontained, the problems can be effectively solved, generation of redundant test data is reduced, premature convergence of an algorithm can be avoided, and the software test efficiency can be improvedto a great extent.

A process for constructing a catalogued nucleic acid library in which the proportional representation of the constituents is adjusted to advantage through the use of disclosed technologies for positive and negative selection. The resultant benefit is that significantly fewer library constituents will need to be screened in order to identify a potentially desired constituent. Moreover, library constituents that previously would have been essentially “lost” are now recoverable. Preferred embodiments of this invention include the cataloguing, normalization, and enrichment of library constituents. By way of example, but not limitation, this technology is serviceable for constructing a library that contains an adequate representation of desirable constituents that (1) are initially found in low-copy numbers within a sample source or (2) originate from an organism that is problematic to culture. Applicable uses of this invention include any library-screening endeavor previously hindered by logistical impediments. By expanding previous logistical frontiers this invention allows for a novel generation of previously unattainable molecules—particularly molecules that are “unclonable” from conventional, unadjusted libraries—to now be detected, cloned, manipulated, expressed, studied, and used. By disclosing the construction and screening of high yielding nucleic acid libraries from mixed and uncultivated organisms, the instant technology eclipses former boundaries in the area of biological discovery and enables the full breadth of biological diversity to be accessed in the search for previously undiscovered genes and gene products. The benefits of the present invention are seen to extend to areas of diagnosis, medicine, agriculture, manufacturing, and academia.

The present invention provides the use of the cell surface antigen CD51 as a negative selection marker for neuronal cells and a method for enrichment, isolation and/or detection of neuronal cells comprising the steps a) contacting a sample containing neuronal cells with an antigen-binding fragment specific for the CD51 antigen coupled to a solid phase, thereby labeling the CD51 positive cells of said sample and b) isolating the non-labeled cells of said sample.

The invention relates to a negative selection marker for escherichia coli gene knock-in, namely, the virulent gene ccdB driven by a plac promoter, and plac-ccdB and the gentamicin resistance gene aacC1 form a gene cassette for gene knock-in. In the first step, DNA fragments containing homologous arms plac-ccdB and aacC1 are obtained through amplification, and electro-transformed into escherichia coli for expression of recombinase. Under gentamicin screening, recombinase catalyzes homologous recombination and the DNA fragments are integrated into escherichia coli genome. In the second step, same homologous arms are utilized for amplification of foreign DNA fragments, ccdB is expressed in the bacterial strains obtained in the first step during the electrotransformation for expression of recombinase under the screening of isopropyl-beta-d-isopropylthiogalactoside, the toxicity serves as the feature of the negative selection marker, recombinase catalyzes homologous recombination among homologous arms, and foreign DNA fragments replace a plac-ccdB-aacC1 gene cassette to realize gene knock-in.

The invention relates to specifically relates to a plasmid vector capable of realizing directional TA cloning, background sticky end-free cloning and expression, and a construction method thereof, belonging to the field of gene engineering. The plasmid vector is a closed circular double-stranded DNA and contains two multiple cloning sites, wherein a ccdB expression cassette exist between the two multiple cloning sites, and the ccdB expression cassette can realize constitutive expression of toxic protein ccdB, which is used as a negative selection marker, in most Escherichia coli strains. The multiple cloning sites are designed on the basis of the plasmid vector; and the plasmid vector is applicable to directional TA cloning and improved background sticky end-free cloning. Moreover, the plasmid vector is directly applicable to expression and purification of proteins. Each function of the plasmid vector is verified. The plasmid vector provided by the invention is expected to play an important role in the field of gene engineering due to its convenience and high efficiency.

Methods and systems for detecting positive, neutral, or negative selection at a locus include obtaining a test sample of cell-free nucleic acids from a subject, preparing a sequencing library of the cell-free nucleic acids, sequencing the library to obtain a plurality of sequence reads, analyzing the sequence reads to detect and quantify one or more somatic mutations at the locus, determining a selection coefficient for the locus, and comparing the selection coefficient with a threshold value to detect positive, neutral, or negative selection at the locus.

It was found that it is possible to construct a genome-edited plant in which no exogenous gene is incorporated in a genome by performing negative selection using morphological defects and the like as indices in a regenerated plant originated from a plant cell in which a gene that induces regeneration of the plant and a gene of the genome editing enzyme are introduced.

The invention discloses an unknown threat detection method based on an artificial immune thought, which is used for effectively detecting known and unknown threats. A pre-classification module introduces a convolutional neural network to solve the problems of pre-collection of a self set and pre-classification of network traffic in detection; a negative selection module introduces a gene bank to solve the random generation problem of an initial detector and the specific immunity problem for unknown threats, and introduces hierarchical clustering to improve the training efficiency of the detector; a cloning variation module is used for solving the overlapping detection problem of a high-affinity detector by introducing a detector optimization algorithm based on a genetic algorithm; meanwhile, an LRU-based memory detector fading mechanism is introduced, so that the storage space is effectively released, and the detection efficiency is improved; and an mRNA vaccination module introduces an mRNA vaccine algorithm based on feature importance sorting, decomposes the detected unknown threats according to gene importance and injects the unknown threats into a gene bank, and generates a corresponding detector to realize specific immunization of the unknown threats and variants thereof.

Described herein are methods and compositions for producing a gene of interest (GOD, which, in certain embodiments, can reduce the metabolic burden on cells and reduce decoupling of GOI production from marker production, as compared to prior art methods. The methods relate to positive selection and negative selection approaches to establishing high GOI-producing cell lines, e.g., CHO lines. In certain embodiments, the methods comprise transfecting a cell with (a) an oligonucleotide comprising a GOI and a non-coding RNA, and (b) an oligonucleotide encoding a selection protein; wherein the non-coding RNA promotes or inhibits production of the selection protein. The cell producing the can be identified and/or selected as a result of or by detecting the absence or the presence of the selection protein.

The present invention provides novel donor polynucleotides formed by linking the two ends of a genomic fragment containing a cleavable site by a polynucleotide carrying a positive selection marker gene and a negative selection marker gene. Use of the donor polynucleotide makes it possible to modify only a target gene with avoiding the possibility of introducing mutations to sequences, called “off-target”, which are other than the target sequence, by introducing cleavage in a homologous site of the donor polynucleotide without introducing cleavage in a target gene locus.

The invention discloses a DNA aptamer of a targeted PD-L1 extracellular fragment, the aptamer is a nucleic acid aptamer PD-L1-Aptamer screened by adopting a Whole-Cell SELEX and a WCS technical screening method, a negative selection cell line PDL-1-KO cancer cell line constructed in the early stage is utilized, and the PD-L1-Aptamer with high specificity and tight combination is screened out through circulation for 20-30 rounds, and the invention relates to the technical field of bioengineering. According to the DNA aptamer of the targeted PD-L1 extracellular fragment, PDL-1 protein expressed on the surface of a cell is taken as a target spot, a corresponding nucleic acid aptamer (PD-L1-Aptamer) is screened out by adopting a whole-cell SELEX technology, and the screened nucleic acid aptamer is subjected to functional research, so that the application of the DNA aptamer in early diagnosis and treatment is developed, in the early stage, a PDL1 gene knockout tumor cell line is constructed by utilizing a crisp-9 technology, the tumor cell line can be used as a very key negative control cell line in the process of screening nucleic acid aptamers, and the purpose of rapidly screening target molecules is achieved by screening PD-L1 positive cells and negative cells and performing bidirectional screening.