17 results about "Cell immortalization" patented technology

The invention belongs to the technical field of biology, and particularly relates to an immortalized alpaca kidney cell line and a construction method and application thereof. The immortalized alpaca kidney cell line disclosed by the invention is named as immortalized alpaca kidney cells (Alpaca kidney cells) BNalpaca 1.0, and the preservation number of the immortalized alpaca kidney cell line is CGMCC (China General Microbiological Culture Collection Center) No.46058. The construction method of the immortalized alpaca kidney cell line comprises the following steps: extracting and culturing alpaca primary kidney cells; infecting alpaca primary kidney cells by using SV40 large T antigen immortalized lentivirus; carrying out resistance screening on the immortalized alpaca kidney cells; carrying out alpaca kidney cell immortalized cell detection and identification; and obtaining the immortalized alpaca kidney cell line. The immortalized alpaca kidney cell constructed by the invention has the advantages of simple culture, repeated passage, good homogeneity, high expression quantity of constructed exogenous genes and the like, and provides a more powerful tool cell for research on alpaca immunity; the immortalized alpaca kidney cell line provides a new tool for virus infection mechanism, drug in-vitro function detection and antibody drug development and screening.

The invention discloses a monoclonal goat intramuscular precursor fat cell immortalized cell line as well as a preparation method and application thereof, and relates to the field of immortalized cells. The method comprises the following steps: acquiring precursor fat cells in primary goat muscle; the method comprises the following steps: infecting precursor adipocytes in primary goat muscle with a lentivirus carrying SV40-T, and adding puromycin to screen positive cells infected with the lentivirus carrying SV40-T; a limited dilution method is used, the screened positive cells are laid on a plate according to one cell per hole, and the monoclonal goat intramuscular precursor fat cell strain is obtained after culture; and carrying out amplification culture on the monoclonal goat intramuscular precursor fat cell strain to obtain the immortalized cell line of the monoclonal goat intramuscular precursor fat cell. According to the immortalized cell line obtained by the method, while the morphology and the function of the precursor fat cells in the goat muscle are ensured, the passage generation number of the cells is remarkably increased, and the cells do not need to be repeatedly sampled and separated, so that the same genetic background of the cells is ensured, and the cost and the workload are greatly saved.

The application discloses an exosome drug delivery system with anti-phagocytosis and tumor targeting ability as well as a preparation method and application thereof. A tumor targeting group is flexibly connected to the outside of the cell membrane of a transmembrane protein with anti-phagocytosis ability through a linker of a fusion protein. A fusion gene sequence is designed and inserted into an expression vector to form a fusion expression vector, which is integrated into the chromosome of a cell to be modified to obtain an engineered cell stably expressing the fusion protein, which is used for secreting engineered exosomes. The cell to be modified includes a naked cell (an immortalized cell line, a primary cell), an engineered cell stably expressing a therapeutic nucleic acid molecule or a protein. The exosomes produced by the former cell can further package the therapeutic molecules to treat tumors, and the exosomes produced by the latter cell can be directly used for treating tumors after being separated, purified and collected. The half-life of the engineered exosomes produced by the application is significantly prolonged in the body, the treatment effect is improved, and the exosomes show strong tumor targeting and anti-tumor effects in vivo and in vitro.

The invention belongs to the technical field of biology, and discloses a porcine testicular mesenchymal stem cell immortalized cell line establishment method and application thereof, and the method comprises the following steps: 1) separating porcine testicular mesenchymal stem cells; (2) enriching the swine testis mesenchymal stem cells; and (3) immortalizing the pig testicular mesenchymal stem cells: inoculating the primary pig testicular mesenchymal stem cells, and transferring SV40 large T antigen into the cells by utilizing lentivirus, so that the pig testicular mesenchymal stem cells are immortalized. The pig testicular mesenchymal stem cell immortalized cell line obtained by the invention is uniform in quality, can be stably passaged for more than 60 generations, is cultured for more than 5 months, and can secrete testosterone under the stimulation of LH (luteinizing hormone). Therefore, the immortalized cell line of the porcine testis mesenchymal stem cells is successfully established, abundant cell resources can be provided for future fundamental research and disease treatment, and a foundation is laid for researching the characteristics of the mesenchymal cell lineage.

A DNA immortalization construct includes a first nucleic acid sequence derived from the 5′ end of human CDKN2A gene exon 2. An EF1α promoter sequence is connected to the first sequence. A first LoxP sequence is connected to the EF1α promoter sequence. A human TERT gene sequence is connected to the first LoxP sequence. A second LoxP sequence is connected to the human TERT gene sequence. An internal ribosomal entrance site (IRES) sequence is connected to the second LoxP sequence. An antibiotic selection gene sequence is connected to the IRES sequence. An SV40 poly-A signal sequence is connected to the antibiotic selection gene sequence. A second nucleic acid sequence is connected to the SV40 poly-A signal sequence and derived from 3′ end of human CDKN2A gene exon 2.

The invention discloses a human immortalized fallopian tube fibroblast strain as well as a construction method and application thereof, and belongs to the technical field of cell immortalization construction. The preparation method comprises the following steps: firstly, separating, culturing and purifying fibroblasts from human fallopian tube tissues by virtue of an enzyme digestion method, identifying markers of the fibroblasts, then introducing a simian virus 40 large T antigen gene into primary fibroblasts by virtue of a lentiviral vector, and screening by virtue of puromycin to obtain a cell strain capable of stably expressing SV40T. Finally, the human immortalized fallopian tube fibroblast which can be stably passaged is constructed. The cell strain has the characteristics of strong multiplication capacity and high hereditary stability, and the cells are not obviously aged along with the increase of the number of generations of the cells, which indicates that the human immortalized fallopian tube fibroblast line is successfully established. According to the invention, the problem that the in-vitro culture life of primary fibroblasts is limited is solved, and a reliable in-vitro cell model is provided for research in the fields of fallopian tube related diseases, ovarian cancer origin, tumor microenvironment and the like.

The present invention relates to an immortalized telocyte cell line, to methods for its preparation which include immortalizing intestine-derived cells labeled with a Foxll -driven reporter gene, and uses of the cell line including for supporting organoid growth.

The invention discloses an immortalized cell line, the preservation information of the immortalized cell line is as follows: the classification name of the immortalized cell line Im-PROS-ADSCs Homo sapiens is PIK3CA related over-growth disease spectrum (PROS), the preservation unit is China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC NO: C2023155, and the preservation date is August 16, 2023. The construction method of the immortalized cell line comprises the following steps: aiming at a clinical symptom of hyperplasia of fat in a PROS disease spectrum, taking facial invasive lipoma as an entrance, extracting adipose tissue-derived stem cells, and transferring SV40LT virus into the adipose tissue-derived stem cells for cell immortalization construction to obtain a first immortalized cell line aiming at the PROS disease. The immortalized cells aiming at PROS disease (facial invasive lipoma) adipose tissue-derived stem cells are established for the first time, the properties of the immortalized cells are researched in vivo and in vitro, the gene expression and drug targets of the immortalized cells are analyzed, an effective research tool is provided for follow-up research, and the immortalized cells have wide application prospects.

The invention discloses a construction method of a yak fallopian tube epithelial cell immortalized cell line, and relates to the technical field of immortalized cell line construction. Two lentivirus packaging recombinant plasmids pLVX-hTERT-Puro and pLVX-SV40LT-Puro are constructed, primary yak fallopian tube epithelial cells are co-transfected, the immortalized yak fallopian tube epithelial cell line is obtained, the cell line has the advantages of being high in growth speed, high in activity, stable in form and capable of being subcultured infinitely in vitro, the cell line has the cancerization-free characteristic, and the cell line can be used for preparing an immortalized yak fallopian tube epithelial cell line. Immortalized genes can be stably expressed, the number of chromosomes is consistent with that of primary cells, and hormone receptors of estrogen and progestational hormone are normally expressed. Therefore, the cell line constructed by the construction method of the yak fallopian tube epithelial cell immortalized cell line is safe and reliable, and a safe and reliable in-vitro model is provided for researching related problems in the field of yak breeding.

A method for extending the replicative lifespan of T cells by activating the TERT gene is provided. Particularly, the method involves the epigenetic modification of the TERT promoter using a CRISPR / dCas9-based epigenetic modifier, followed by the reactivation of the modified TERT promoter using a Cas9-mediated transcriptional activator.

The invention discloses a Putian subpigskin precursor fat cell immortalized cell line, a preparation method and application, and relates to the technical field of immortalized cells, and the method is characterized in that the immortalized Putian subpigskin precursor fat cell line is established by using an SV40T lentivirus infection system; the differentiation capacity of precursor fat cells is continuously reserved, the tumorigenesis tendency does not occur, and the cell line belongs to a normal cell line. The cell line not only can provide a stable breed conservation material for protecting genetic resources of Putian pigs, but also is beneficial to providing a reliable biological material for exploring a pig fat deposition molecular mechanism.

The invention discloses an establishment method of a naturally immortalized pilose antler regeneration bud base cell line and an engineering preparation method of functional factors of the naturally immortalized pilose antler regeneration bud base cell line, and belongs to the technical field of biology. The method comprises the following steps: obtaining pilose antler regeneration bud bases, performing primary culture and identification on regeneration bud base cells, screening to obtain dominant growth cell strains, performing engineering preparation and purification on secretion factors of the regeneration bud base cells by adopting a cell culture bioreactor, and identifying the activity of the factors. According to the method, the pilose antler regeneration bud cell line which is high in activity, high in proliferation rate, capable of retaining bud cell attributes and capable of being subcultured for at least 200 times is preferably selected, so that the natural immortalization of the cells is realized, and the biological characteristics of the immortalization of the pilose antler regeneration bud cell are identified. Meanwhile, the problems that the pilose antler regenerated bud-based cells cannot be directly applied to the safety and ethics of a human body and are high in cost and difficult to produce on a large scale are solved by a method for producing functional factors, and the pilose antler regenerated bud-based cell secretion factors with biological activity are prepared in an engineering manner and purified.

Provided are a B-cell immortalization-based method for developing a single B-cell antibody and the use thereof. The provided method comprises the following steps: S1, antigen preparation; S2, antigen immunization; S3, modification of a lentiviral vector; S4, viral infection and cell sorting; S5, screening for cells with a high binding activity; S6, sequencing of an antibody sequence; S7, activity verification of a target antibody, etc. The provided antibody development method effectively solves the problems that single B-cell antibody development requires extensive recombinant expression and incurs a high verification cost. By combining single B-cell technology with B-cell immortalization technology, a B cell is infected by using a modified lentivirus, so that the cell achieves immortalization and enables in vitro proliferation and antibody secretion. Furthermore, by combining high-throughput microfluidic screening with cell immortalization technology, cell infection is performed directly in an oil droplet, thereby achieving high-throughput and low-cost antibody development.

The invention discloses a construction method and application of a matrigel suppository telocyte immortalized cell line, which comprises the following steps: recruiting in vivo matrigel suppository, sorting telocytes by CD34 immunomagnetic beads, and combining SV40T transduction to establish the telocyte immortalized cell line. And carrying out morphological observation, specific marker detection and functional verification to identify that the obtained cell line is the teterocyte. According to the construction method, the teterocyte closer to the in-vivo functional state can be efficiently obtained, and the obtained cell line can be stably passaged for a long time and maintain the morphological characteristics and biological functions of primary cells; a stable and reliable tool is provided for in-vitro research such as subcutaneous microenvironment research, drug screening and cell therapy, tissue engineering and regenerative medicine and continuous in-depth research in related fields, and the method has wide application value in multiple scientific research fields such as tissue engineering model construction, intercellular interaction research and drug effect evaluation.

The present invention specifically relates to a method for developing single B cell antibodies based on B cell immortalization and its application. The method provided by the present invention includes the following steps: S1, preparation of antigens; S2, antigen immunization; S3, modification of lentiviral vectors; S4, virus infection and cell sorting; S5, screening of cells with high binding activity; S6, antibody sequence sequencing; S7, activity verification of target antibodies and other steps. The present invention provides a brand-new method for antibody development, effectively solving the problems of the need for a large amount of recombinant expression and high verification cost in the development of single B cell antibodies. By combining single B cell technology with B cell immortalization technology, the modified lentivirus is used to infect B cells to immortalize the cells, enabling them to proliferate in vitro and secrete antibodies. Secondly, by combining high-throughput microfluidic screening and cell immortalization technology, cell infection is directly carried out in oil droplets, achieving high-throughput and low-cost antibody development.

Disclosed herein are methods and compositions for immortalization of animal cells. Immortal animal cells may have genome modifications to increase proliferative capacity, 3D growth and / or suspension growth. Immortal animal cells with highly proliferative capacity may allow production of meats ex vivo. Such production of meat may lessen food chain burdens or tighten nutrition control of the meat produced.

The purpose of the present invention is to provide a method for causing cell immortalization of an antibody, and an immortalized transformed cell obtained by the method. The present invention relates to: a method for producing an immortalized cell by introducing, into a non-immortalized antibody-producing cell, an SV40T antigen gene, a Bcl-2 family gene, and one or more genes selected from the group consisting of a Myc family gene and a Cyclin D family gene; the invention also relates to a manufacturing method of the immortalized antibody producing cell. An immortalized antibody-producing cell is produced by collecting an antibody-producing cell from a mammal, performing primary culture, and introducing, into the obtained primary antibody-producing cell, an SV40T antigen gene, a Bcl-2 family gene, and one or more genes selected from the group consisting of a Myc family gene and a Cyclin D family gene.