70 results about "Piggybac transposon" patented technology

Disclosed herein are compositions comprising integrating enzymes that can deliver nucleic acids to a target DNA. Additionally, the methods of using the compositions disclosed herein relate to treatments for a variety of infections, conditions, and genetic disorders.

The present invention provides hyperactive piggyBac transposons, in particular hyperactive piggyBac transposons from Trichoplusia ni (cabbage looper moth) that transpose at a higher frequency than wildtype. The invention also features integration defective piggyBac transposons. The piggyBac transposons and transposases can be used in gene transfer systems for stably introducing nucleic acids into the DNA of a cell. The gene transfer system can be used in methods, for example, but not limited to, gene therapy, insertional mutagenesis, or gene discovery.

PiggyBac transposons and transposases with enhanced transposition activity in cells are provided. Also provided are associated methods and kits for both introducing exogenous DNA inserts into the genomes of host cells as well as for the removal of the inserts from the host cell genomes. Cells obtained by use of the compositions, methods and kits are also provided.

The piggyBac transposon is disclosed herein as an extremely versatile helper-dependent vector for gene transfer and germ line transformation in a wide range of vertebrate species. Presented are methods wherein genome sequencing databases may be examined using piggyBac, as homologues of piggyBac have been found among several sequenced animal genomes, including the human genome. This transposon is demonstrated to provide transposition in primate cells and embryos of the zebra fish, Danio rerio. PiggyBac mobility is demonstrated using an interplasmid transposition assay that consistently predicts the germ line transformation capabilities of this mobile element in several species. Both transfected COS-7 primate cells and injected zebrafish embryos supported the helper-dependent movement of tagged piggyBac element between plasmids in a cut-and-paste, TTAA target-site specific manner. The present invention discloses the use of piggyBac as a tool for genetic analysis of vertebrates.

The present invention is directed to a transformation system for making transgenic organisms that includes a vector containing a modified piggyBac transposon into which is inserted an enhanced green fluorescent protein gene linked to a polyubiquitin promoter sequence and a nuclear localizing sequence; and a helper transposase vector that includes an hsp7O promoter sequence upstream of the putative piggyBac promoter that increases the transformation frequency of this system.

The invention discloses a method for synthetizing secretion lysozyme by middle silkgland cell of silkworm. The method comprises the following steps: building a pBSer1hLYZ (lysozyme)-A3EGFP (Enhanced Green Fluorescent Protein) plasmid for synthetizing secretion lysozyme by the silkworm; then, introducing the plasmid and an assistant plasmid capable of providing transposase into a silkworm germ cell according to the microinjection transgenosis silkworm technology; according to the transposition characteristics of a piggyBac transposon, introducing a green fluorescent protein gene and a lysozymegene into a silkworm gene group, to obtain stable heredity and expression so as to create the transgenosis silkworm capable of synthetizing secretion lysozyme by middle silkgland cell of silkworm by specificity; further, hybridizing the transgenosis silkworm with sericin silkworm; carrying out back crossing on the hybridized descendant with the sericin silkworm for 3-5 generations; and finally, carrying out selfing on the obtained product to carry out homozygosis on the lysozyme gene so as to obtain the new species of the transgenosis silkworm of the secretion lysozyme. According to the method, a basis for improving lysozyme production efficiency and lowering production cost is laid.

The invention discloses a method for building a domestic silkworm silk gland biologic plant. A recombinant transgene vector which is provided with domestic silkworm silk protein specificity promoter controlled extraneous gene expression cassettes, an enhancer activity component, antibiotic resistant genes and fluorescent protein reporter genes and based on a piggyBAC transposon is established through gene engineering operations. The transgene vector is introduced into primarily laid eggs, special antibiotics are fed to hatched newly exuviated silkworms continuously until the fourth instar. In this way, silkworms which can resist the specific antibiotics and are provided with fluorescent protein reporter genes are obtained; target genes are detected; and transgene silkworms are manufactured by breeding. By adopting the silk gland tissues of the transgene silkworms, protein drugs can be obtained. With the method disclosed by the invention, transgene domestic silkworm silk gland plants can prepare protein drugs at low manufacture cost without potentially hazardous rhabdovirus. The method is safe and is practically valuable.

The invention discloses a method for synthesizing and secreting black widow spider dragline silk protein 1 through a bombyx mori silk gland bioreactor. The method includes the steps that firstly, pBac[3xP3-DsRed]-MaSp1 plasmids serving as a carrier for bombyx mori to synthesize and secrete black widow spider dragline silk protein 1 are established; then, plasmids and assistant plasmids are introduced into bombyx mori zygotes through microinjection, red fluorescence protein genes and black widow spider dragline silk protein 1 genes are introduced into bombyx mori genomes through the transposition characteristic of piggyBac transposons, stable inheritance and expression are performed, transgenic bombyx mori is obtained, mori moth selfing is performed to achieve homozygosis of black widow spider dragline silk protein 1 genes, and transgenic bombyx mori secreting black widow spider dragline silk protein 1 is bred. According to the method, transgenic bombyx mori is screened through fluorescence marker genes, and black widow spider dragline silk protein 1 is specifically synthesized and secreted through bombyx mori silk gland cells; the method is used for development and utilization of spider silk and can also be used for improving the mechanical performance of silk.

The present invention is directed to a transformation system for making transgenic organisms that includes a vector containing a modified piggyBac transposon into which is inserted an enhanced green fluorescent protein gene linked to a polyubiquitin promoter sequence and a nuclear localizing sequence; and a helper transposase vector that includes an hsp70 promoter sequence upstream of the putative piggyBac promoter that increases the transformation frequency of this system.

The invention discloses a compound type piggyBac recombinant vector as well as a preparation method and an application of the compound type piggyBac recombinant vector. According to the recombinant vector, a target gene expression frame, selection marker gene expression frames I and II, piggyBac transposase expression frame regulated by a heat shock promoter and truncated type piggyBac transposon arms L2 and R2 are inserted between wild type piggyBac transposon arms R1 and L1, integration of target gene, selection marker gene, piggyBac transposase expression frame sequence in silkworm genome can be conveniently mediated, complete deletion of all of transposon sequences and selection marker gene sequences in progeny transgenosis silkworm genome can be realized through establishing and screening a transgenosis silkworm line with high target gene expression and then inducing piggyBac to express in the transgenosis silkworm by heat shock treatment, replacement of other exogenous gene and the target gene can be conveniently realized through box type exchange reaction mediated by phiC31 integrase by surplus target gene expression frame anchored by attP locus, and then fixed point integration of other exogenous genes in the genome locus is further realized.

The invention belongs to the field of genetic engineering, and specifically relates to a method of domestic silkworm transgene for controlling domestic silkworm pupal stage growth through UAS / GAL4 dual system. According to the method, piggyBAC transposon vector pigA3GFP is adopted as a base vector; through genetic engineering operation, GAL4 gene expressed transgenic domestic silkworm PDP-GAL4 system controlled by domestic silkworm cocoon protease gene promoter (PDP) is established; through genetic engineering operation, domestic silkworm baculovirus egt gene or charybdotoxin BmKIT3R gene expressed transgenic domestic silkworm UAS-egt or UAS-BmKIT3R system controlled by UAS promoter is established; the PDP-GAL4 system and the UAS-egt or UAS-BmKIT3R system are hybridized, such that larval stage growth of an F1 generation is normal, and pupal stage of the F1 generation is prolonged for 7 to 10 days. According to the invention, with an UAS / GAL4 dual system, the F1 generation silkworm pupal stage is prolonged by using domestic silkworm baculovirus egt gene or BmKIT3R gene. Flexibilities of silkworm cocoon purchasing and filature technology are improved. Therefore, the invention has important application value.

The invention discloses a method for producing transgene silkworms for improving resistance to nuclear polyhedrosis. A transgene vector which carries dsRNA expression cassette of ie-1 genes of bombyx mori nuclear polyhedrosis viruses, dsRNA expression cassette of lef-1 genes, is provided with neomycin resistance genes Neo and fluorescent protein reporter genes, and based on a piggyBAC transposon, is established through gene engineering operations. The transgene vector is introduced into primarily laid eggs, and the resistance of the transgene silkworms to nuclear polyhedrosis is significantly improved. By combing transgenes with RNAi technology, the resistance of silkworms to nuclear polyhedrosis is improved, and the screening process of transgene silkworms is optimized.

The invention discloses a reconstructed human serum albumin gene suitable for cultivated silk gland expression and an expression system and application thereof. The reconstructed human serum albumin gene is as shown as the 7-1767th in SEQ ID NO.1, and the coded amino acid is as shown in SEQ ID NO.2. The reconstructed human serum albumin gene, a secretion type sericin 1 gene promoter and a secretion type sericin 1 gene terminator form an expression frame, an enhancer Hr3 is used for enhancing expression, and simultaneously a piggyBac transposon and a fluorescent screening marker gene construct an expression system. The expression system can highly effectively express recombined human serum albumin in cultivated silk gland, has biological activity, can be used for large scale production of recombined human serum albumin, and has good market prospect.

The invention discloses an expression vector for a piggyBac transposon and a transgenic pig and a construction method thereof, belonging to the field of gene engineering. The expression vector is constructed by connecting a fusion fragment containing a loxP-Neo / EGFP gene expression cassette to pCyL50 plasmid. The vector is introduced into the genome of a pig, and somatic cell nuclear transfer technology is employed to produce the transgenic pig. The expression vector provided by the invention has the piggyback transposon, greatly improves transformation efficiency, changes a common plasmid series recombination transgene integration model into a single-site single-copy integration model and better simulates the internal environment of a biological gene. The construction method for the transgenic pig provides a novel approach for preparation of a variety of transgenic animals and a simple, convenient and highly efficient approach for fabrication of biological models of transgenic animals.

The invention discloses application of a phiC31 recombinase system and a piggyBac transposon and a fixed point transgenetic system of silkworm and a preparation method of the fixed point transgenetic system. According to the invention, the phiC31 recombinase system and the transgenic technology for mediating the silkworm to transform by the piggyBac transposon are combined; first, two attB sites or attP sites which are arranged in the same direction are connected into a piggyBac transposon carrier; the carrier in transformed into silkworm; then, single copy transgenetic silkwork is screened to be used as a target system; then, the target system is transformed by using the phiC31 recombinase and a carrier, wherein target gene expression frames are anchored at the two ends of the carrier by the attB sites or attP sites which are arranged in the same direction; and thus the target gene expression frames are transformed to the place between fixed points, namely the two attB sites or attP sites of the piggyBac transposon carrier, thereby realizing the purpose of fixed point transgene of silkwork. The method is simple to operate and provides a powerful tool for genetic function research for silkworm.

The present invention is directed to a transformation systems and vectors for making transgenic organisms that includes a vector containing a modified piggyBac transposon into which is inserted at least one fluorescent protein gene linked to a polyubiquitin promoter sequence. A helper transposase vector that includes an hsp70 promoter sequence upstream of the putative piggyBac promoter that increases the transformation frequency of this system can also be included.

The invention belongs to the field of genetic engineering, and in particular relates to a method for improving egg laying amount of an original strain of mulberry silkworm. The method comprises the following steps: by using a piggyBAC transposons carrier containing a fluorescent protein reporter gene as a basic carrier, constructing a transgenetic mulberry silkworm vlg-GAL4 system or a nanos-GAL4 system which controls GAL4 genetic expression through a vasa-like genetic promoter or a nanos-like genetic promoter through operation of genetic engineering; constructing a transgenetic mulberry silkworm UAS-ovo-1 system which controls genetic expression of mulberry silkworm ovo-1 through a UAS promoter through operation of genetic engineering; hybridizing the vlg-GAL4 system or nanos-GAL4 system with the UAS-ovo-1 system to obtain first-filial generation mulberry silkworm; and selfing and laying by the mulberry silkworm obtained, so that the egg laying amount of original strain of mulberry silkworm is improved. The method provided by the invention over-expresses mulberry silkworm ovo-1 genes in the genital gland through the GAL4 / UAS binary system, so that the laying level of a single moth is improved, the laying amount is increased by over 10%, and meanwhile, the production cost of cocoons is lowered. The method has important application value.

The invention discloses a method for breeding Macropodus opercularis with a transferred green fluorescent protein gene by using piggyBac transposons, which comprises the following steps of: firstly, constructing piggyBac transposons with green fluorescent protein genes and a helper plasmid capable of providing transposase; and secondly, transferring the two transposons into a germ cell of the Macropodus opercularis by using microinjection technology, and according to the transposons characteristics of the piggyBac transposons, introducing the green fluorescent protein genes into a genome of the Macropodus opercularis so as to make the gene inherited and expressed stably. An individual of transgenic Macropodus opercularis can be screened by means of the fluorescence-labeled gene, and the Macropodus opercularis with the transferred green fluorescent protein gene is more worthy of appreciation and represents a new variety of Macropodus opercularis with specific function. In addition, it is the first time for the piggyBac transposons to be used in researches on transgenic fish, and thus, the research space for transgenic aquatic animals is widened.

The invention discloses an efficient transgenosis method with mediation of a transcription activator-like effector (TALE) protein. The method comprises the steps that a helper plasmid containing a transcription activator-like effector protein DNA (Deoxyribonucleic Acid) sequence and piggyBac transposon (PBase) DNA sequence is constructed by adopting a molecular biological technique, and transcribed to mRNA (Messenger Ribonucleic Acid) in vitro; mRNA of the helper plasmid and DNA of a piggyBac transposon plasmid containing an exogenous gene are imported to a fertilized egg of an animal by adopting a microinjection method; a transgenic animal of which the exogenous gene can be stably inherited and expresses is obtained; and a transgenosis positive individual is screened out according to a marker gene or the expression characteristic of the gene. With the adoption of the method, a piggyBac transposon can be efficiently inserted into a genome of a host organism by the action of a TALE-PBase fusion protein; the transgenosis efficiency mediated by the piggyBac transposon can be improved remarkably; and helps are offered to researches such as transgenic animal acquirement and insertion mutagenesis.

The present invention relates to an improved PB transposon system and application thereof. Specifically, the present invention relates to a nucleic acid construct. The nucleic acid construct sequentially contains a promoter for controlling the expression of PiggyBac transposase, a PiggyBac transposase coding sequence, a terminal repeat at the 5'-end of the PiggyBac transposon, an optional polyA tailing signal sequence 1, a polyclonal insertion site, a polyA tailing signal sequence 2 and a terminal repeat at the 3'-end of the PiggyBac transposon. A recombinant vector constructed by using the nucleic acid construct provided by the invention has obviously improved integration efficiency.

The invention relates to a novel genetically modified plasmid vector, its construction and a cultivation method of silkworms containing a novel recombination gene in vivo. The novel genetically modified silkworm has a recombinant fibroin heavy chain protein gene containing MMPs enzyme sites. The invention belongs to the field of agricultural insect excellent variety cultivation and utilization. A flow diagram for construction of the novel genetically modified vector is as shown in the attached map. By the characteristic that the MMPs enzyme sites can undergo MMPs enzyme digestion, the MMPs enzyme sites are introduced into silkworm fibroin. Thus, the silkworm fibroin has new properties and the purpose of modifying silkworm fibroin through a biological method is achieved. According to the patent, the recombinant fibroin protein gene is cloned to a PiggyBac transposon vector; the vector is injected into silkworm eggs through a micro-injection method; after incubation of the silkworm eggs, individuals with green fluorescence eyes are selected so as to determine the successfully-cultivated novel genetically modified silkworms.

The invention belongs to the field of biotechnology, and relates to a resource library of rat strains with gene mutation and a preparation method thereof. The rats with gene mutation in the resource library can be obtained from rat spermatogonial stem cells, embryonic stem cells or fertilized ovum with piggyBac transposon insertion mutation. The rats with gene mutation in the resource library also can be obtained from shifting the transposon in germ cells of male rats with mutation. The library can be applied to animal phenotype analysis, gene function research, human disease simulation, medicament screening and drug target development.

The invention discloses a construction method of a piggyBac transposon vector for producing a transgenic goat. The construction method mainly comprises the steps of: (1) synthesizing a section of base sequence comprising a PB5' end and a PB3' end necessary for transposition of a piggyBac transposon, reserving multiple cloning sites in the middle of the PB5' end and the PB3' end during synthesis, and then cloning the sequence into a pBluSKm vector to construct a pBluSKm-PB vector; and then cloning a rumen-specific expression promoter PRD-SPRR II, cysteine synthesis genes cysE and cysM which are subjected to codon preferred optimization, a Neomycin resistant gene and a green fluorescent protein gene to the pBluSKm-PB vector to finally construct a pBluSKm-PB-PRD-cysEM-NEO-EGFP transposon vector. The transposon vector obtained by the invention can meet the requirements of cell screening, micromanipulation under an inverted fluorescence microscope, production of the transgenic goat and the like.

The invention discloses a method for synthesizing and secreting black widow spider traction silk protein 2 through a silkworm silkgland bioreactor. The method comprises the steps that a pBac[3xP3-DsRed]-MaSp2 plasmid carrier of the black widow spider traction silk protein 2 synthesized and secreted by silkworms is built, then the plasmid and auxiliary plasmid are introduced into silkworm fertilized ova through micro-injection, red fluorescence protein genes and black widow spider traction silk protein 2 are introduced into silkworm gene groups through transposition characteristics of a piggyBac transposon, inheritance and expression are stabilized, transgenic silkworms are made, silkworm moth selfing is carried out to achieve homozygosis of black widow spider traction silk protein 2 genes, and the transgenic silkworms for secreting black widow spider traction silk protein 2 are bred. The transgenic silkworms are screened by means of fluorescence marker genes, the black widow spider traction silk protein 2 is specifically synthesized through the silkworm silkgland cells for spider silk development and utilization, and meanwhile the mechanical performance of silk is improved.

The invention belongs to the technical field of gene engineering, and particularly relates to a transposon carrier and system for preparing an immortalized cell and a usage method thereof. The transposon carrier and system for preparing the immortalized cell and the usage method thereof aim at solving the technical problem of providing a new choice for preparing the immortalized cell. According to the technical scheme, the piggyBac transposon carrier for preparing the immortalized cell contains an SV40TAg, the two ends of the SV40TAg are provided with FRT sites, and the two ends of a fragment (hygromycin-SV40TAg or neomycin-SV40TAg) which is actually inserted into a genome contain piggyBac transposase splice sites. The invention further provides a host cell containing the carrier and the system for preparing the immortalized cell, and the system comprises the carrier and a piggyBac transposase expression carrier. By adopting the carrier, an immortalized cell system can be established more effectively and quickly.

The invention provides a transgenic vector, a method for rapidly constructing an ACE2 humanized animal model by using the transgenic vector, and application of the ACE2 humanized animal model by aiming at research and development of SARS-CoV-2 drugs. The transgenic vector comprises a PiggyBac transposon 5'end inverted repeat sequence (ITR), a CAG promoter, a human ACE2 coding region, a ribosome access site (IRES), firefly luciferase, a dial rat hepatitis post-transcriptional regulatory element (WPR), a polyA site and a 3 'end inverted repeat sequence (ITR), the transgenic vector can efficiently insert human ACE2 and luciferase gene expression cassettes into a mouse genome, and the luciferase and human ACE2 expressed transgenic mice can be rapidly screened through a luciferase living body imaging system.

The invention discloses a method for obtaining stable cell populations for expressing target protein in CHO cells through piggyBac transposons. The method includes the steps that 1) plasmid of gene segments containing to-be-expressed coding target protein genes and antibiotic screening genes and plasmid containing piggyBac transposons sequences are established; 2) the plasmid established in the step 1) is transfected and led into the CHO cells at the same time; 3) the cells after being transfected in the step 2) are directly subjected to antibiotic screening in a shake flask. By means of the method, the stable cell populations can be established in one week to two weeks, and the target protein can be efficiently expressed. The invention also discloses a method for obtaining the stable cell populations for expressing the target protein in the CHO cells through the piggyBac transposons and directly producing the target protein in an enlarged mode, and production of 50 liters or above can be met.

The invention relates to a bombyx mori whole genome knockout vector library based on a CRISPR / Cas9 system and a construction method. The vector library comprises a delivery vector system piggyBac transposon vector system used for delivering a CRISPR / Cas9 system and a vector library pB-CRISPR-library composed of sgRNA sequences of targeting sites of genes of all encoding proteins of bombyx mori. According to the invention, based on the strong bearing capacity of the piggyBac transposon system, a Cas9 protein expression cassette and an sgRNA expression cassette, which are two constituent elements of a CRISPR / Cas9 system, can be integrated into one vector; and meanwhile, the vector further comprises a screening marker Zeocin resistance gene expression cassette, and the two parts jointly forman all-in-one vector pB-CRISPR for CRISPR / Cas9 knockout of bombyx mori.

The invention relates to a spider MaSps gene and an application of the spider MaSps gene in preparation of drug-loaded nanoparticles. The artificially designed and modified optimized spider MaSps geneis disclosed for the first time; a transgenic expression cassette in which fusion expression of a silk fibroin heavy chain protein (Fib-H) gene and a spider MaSps gene is regulated and controlled bya bombyx mori silk fibroin heavy chain promoter is constructed; a piggyBac transposon arm and a fluorescent selection marker gene are connected to construct a transgenic expression vector; the expression vector can be used for establishing transgenic bombyx mori capable of efficiently expressing recombinant fusion MaSps protein in bombyx mori silk glands, so that transgenic cocoon silk containingthe recombinant fusion MaSps protein in silk fibroin is obtained; and further a transgenic silk material which can stably secrete the recombinant MaSps protein in a silk fibroin layer and has the effects of promoting controlled release of drugs and endocytosis is obtained.