47 results about "Puromycine" patented technology

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 present invention describes a composition comprising an effective amount of a combination of two or more components selected from acacetin, ACT1 peptide, α-lipoic acid, alprostadil, anisomycin, apigenin, ascorbic acid, astragalus, berberine, β-lapachone, β-hydroxy-β-methylbutyrate, bacopa monnieri, catechins, catechols, chamomile, chrysin, coumestrol, curcumin, dinitrophenol, dinoprost, ellagic acid, (-)-epigallocatechin gallate, green tea extract, fisetin, genistein, ginsenoside RE, glabridin, 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid, glycyrrhizin, hydroquinone, isoquercetin (EMIQ), shan Phosphate, black bean anthocyanins, leucine, lithium, luteolin, luteolin, luteolin, melatonin, menadione, 1‑methylnicotinamide (MNA), methyl salicylate, myricetin, nicotinamide adenine dinucleotide, nicotinic acid (vitamin B3), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide (NaAD), nicotinic acid mononucleotide (NaMN), parsley, phenylephrine, pokeweed mitogen, 15‑Δ prostaglandin J2, puromycin, quercetin, quinolinic acid, retinoic acid, trichostatin A, troxerutin, rutin, tryptophan, vitamin D3, withaferin A, wortmannin, and zinc (including its salts).

The invention discloses an SOD2 knockout cell line and application of the SOD2 knockout cell line in anti-poxvirus, and relates to the technical field of antiviral research. According to the invention, sgRNA of targeted superoxide dismutase 2 (SOD2) is designed, the sequence of the sgRNA is shown as SEQ ID NO.1-SEQ ID NO.2, then a stable SOD2 knockout human-derived non-small cell lung cancer A549 cell line is established by combining CRISPR / Cas9 gene editing with a lentiviral vector delivery technology, single-cell cloning is obtained through multiple rounds of puromycin screening, and the SOD2 gene knockout A549 cell strain is successfully constructed. Vaccinia virus Tian Tan strain infection shows that the number of plaques of SOD2 knockout cells is obviously greater than that of the plaques (about 2.3 times) of normal cell infection, and the plaques are relatively large, so that fine SOD2 has the effect of limiting intercellular transmission of poxvirus. The invention lays a foundation for research and preparation of antiviral drugs.

The invention discloses a yak intramuscular precursor fat immortalized cell line and a preparation method thereof. The method comprises the following steps: (1) obtaining yak primary intramuscular precursor fat cells; (2) performing SV40T lentivirus infection on the precursor fat cells in the yak muscle and screening cell strains: performing SV40T lentivirus infection by utilizing a 1 / 2 small-volume infection method to obtain immortalized cells after the precursor fat cells in the yak primary muscle are infected with SV40T, namely immortalized cells for short; puromycin is adopted to screen immortalized cells; and diluting the screened cells by using a limited dilution method, and carrying out enlarged culture to obtain the immortalized cell line. The yak intramuscular precursor fat immortalized cell line constructed by the invention has genetic homogeneity, the heterogeneity influence of cell populations is eliminated, and an in-vitro cell model is provided for subsequent research related to yak intramuscular fat cells.

The invention relates to the technical field of gene engineering, and relates to a construction method of sgRNA with a pBD2 gene knocked out and a construction method of a pig intestine epithelial cell line with the pBD2 gene knocked out. According to a pBD2 gene sequence, sgRNA capable of targeting the gene is designed and synthesized, plasmids of a CRISPR / Cas9 system containing the sgRNA are constructed by using a molecular cloning and recombination technology, the plasmids are transfected into a pig small intestine epithelial cell line, gene editing is performed on a specific position of the pBD2 gene, and a stable monoclonal cell line with the pBD2 gene knocked out is obtained through puromycin drug screening. The homozygous pBD2 gene knockout cell strain obtained by the method is used as a cell model and can be used for exploring the effects of the pBD2 gene in the aspects of immunoregulation, sphingolipid metabolism, cell cycle and the like of small intestine cells.

The invention provides a construction method of a non-small cell lung cancer histone H1.3 arginine methylation point mutation cell model. The construction method comprises the following steps: designing mutation primers H1.3 R80A-F and H1.3 R80A-R; the method comprises the following steps: by taking a pCDH-HA-H1.3-Flag plasmid as a template, carrying out PCR (Polymerase Chain Reaction) amplification by adopting a mutation primer, digesting a product by DMT enzyme, and converting a competent cell to obtain a mutant plasmid; and co-transfecting the mutant plasmid and a helper plasmid to a packaging cell, collecting a virus solution, filtering, infecting an A549 cell, adding Polybrene to assist infection, culturing, and screening a stably transfected cell strain by using puromycin to obtain the recombinant plasmid. The invention also provides application of the mutant cell model obtained by the construction method. According to the invention, the blank of histone H1.3 methylation research is filled, the 80th arginine is clear as a core modification site, and the established model provides a new tool for lung cancer mechanism research and drug research and development.

The invention belongs to the technical field of gene engineering, and particularly relates to a construction method and application of a recombinant vector based on bCoro1a gene 3 'UTR editing. The recombinant vector consists of an upstream homologous arm of a targeting site, 3 'UTR inserted into a target sequence, 200bp of a nucleotide tailing signal of bCoro1a, a selection marker and a downstream homologous arm of the targeting site; the targeting site is the 29 or 70 site of the 3 'UTR of the bCoro1a gene; the sequence of the 3 'UTR of the bCoro1a gene is as shown in SEQ ID NO. 48; the selection marker comprises enhanced green fluorescent protein and puromycin, the bCoro1a 3 'UTR edited gene expression regulation and control system is based on bCoro1a 3' UTR edited gene expression regulation and control system, and the expression level of bCoronin-1A is reduced in a targeted manner by inserting a specific bta-miR-27b target sequence into 3 'UTR, so that M.tb immune escape is inhibited, and the M.tb infection resistance of a host is improved.

The invention discloses a homologous recombination system, a transposition system, a fruit fly animal gene high-throughput screening method and application. The screening method comprises the following steps: firstly, separating and establishing a stable cell line from a fruit fly animal; then co-transfecting a CRISPR / Cas9 mediated homologous recombination system, and obtaining a cell line for stably expressing Cas9 protein through puromycin screening and a limited dilution method; the sgRNAs plasmid library constructed by combining a PiggyBac transposon system can realize efficient screening and functional analysis of a target gene in the Cas9 cell line. The high-throughput screening method provided by the invention is widely applicable to gene function research and target gene screening, is simple in preparation process and convenient to operate, has good universality, and provides effective technical support and solution for related fields.

The invention belongs to the field of gene engineering, and particularly relates to construction of an RNF31 gene knockout cell line and application of the RNF31 gene knockout cell line as a foot-and-mouth disease virus vaccine production cell line. The method comprises the following steps: firstly, designing two sgRNA sequences at an RNF31 exon by utilizing a CRISPR / Cas9 technology, and connecting with a pX459-puro vector to construct a recombinant plasmid; the method comprises the following steps: transfecting a PK-15 cell with pX459-RNF31-sgRNA, and screening under the action of puromycin, so as to obtain the RNF31 gene knockout cell line. Compared with a wild type cell, the RNF31 gene knockout cell line remarkably promotes FMDV replication and can be used as a production cell line of a foot and mouth disease virus vaccine; and data support is provided for further researching a mechanism of inhibiting FMDV replication by RNF31.

The invention relates to the field of biomedical research, and provides a lung cancer cell strain (H1299) capable of stably and low-expressing Integrin alpha6 protein and a construction method of the lung cancer cell strain (H1299). The preparation method comprises the following steps: designing and synthesizing an shRNA (short hairpin Ribonucleic Acid) sequence for specifically knocking down gene expression aiming at a human Integrin alpha6 gene, forming a double-chain fragment through annealing, and inserting the shRNA sequence into a pLKO.1-puro lentiviral vector by adopting a connection independent cloning (LIC) method, so as to construct a pLKO.1-ITG alpha6-shRNA recombinant plasmid. Furthermore, the recombinant plasmid and lentivirus packaging plasmids psPAX2 and pVSVG are co-transfected to an HEK293T cell by utilizing a lentivirus packaging system, and the lentivirus is produced by packaging. And infecting a target lung cancer cell strain H1299 by using the obtained lentivirus, and then carrying out puromycin resistance screening and continuous subculture to finally obtain the stable and continuous low-expression Integrin alpha6 protein H1299 cell strain. A fluorescence microscope, a real-time fluorescent quantitative PCR (Polymerase Chain Reaction) and an immunoblotting technology are used for verifying that the gene silencing efficiency of the Integrin alpha6 in the cell strain can reach more than 80%. The stable cell strain constructed by the invention provides a reliable and efficient tool cell model for researching the molecular mechanism of the Integrin alpha6 in the processes of occurrence and development, invasion and metastasis, energy metabolism, immune escape and the like of lung cancer, and also provides a new experimental material for screening antitumor drugs targeting the Integrin alpha6.

The invention provides a low-autophagy Vero cell line based on a DDIT3 inhibition gene as well as a construction method and application of the low-autophagy Vero cell line. The cell line stably knocks down DDIT3 expression through lentivirus-mediated shRNA, the autophagy level under the condition of nutrition deficiency is remarkably reduced, the cell proliferation capacity is enhanced, and the yield of influenza A H1N1 viruses is remarkably increased. The construction method comprises the following steps: cloning shRNA (short hairpin ribonucleic acid) to a GV493 lentiviral vector, co-transfecting 293T cell packaging virus with psPAX2 / pMD2. G helper plasmid, infecting Vero cells, and screening through puromycin to obtain a stable cell line. The cell line solves the problem of growth stagnation caused by over-activation of autophagy in serum-free suspension culture of traditional Vero cells, and is suitable for large-scale vaccine production.

The present invention provides a single-plasmid inducible expression system and its preparation method and application, which belong to the field of genetic engineering technology. The single-plasmid inducible expression system comprises a single plasmid, and the nucleic acid sequence of the plasmid is shown in SEQ ID No. 1. The single-plasmid inducible expression system constructed by the present invention overcomes the complex operation of twice-establishing the system of traditional induced expression. Only one gene transduction is required to screen and obtain the expected transgenic cell line, which simplifies the operation process and improves efficiency. Puromycin can be used for clone screening after gene transduction, and the tetracycline antibiotic doxycycline is used to induce target gene expression. Compared with the lentiviral system, it is simple to operate, saves costs, can insert a larger exogenous gene length, and has a larger vector capacity; the background expression is lower, the induced expression multiple is higher, and efficient gene expression can be achieved while having lower background leakage.

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 invention provides a PK-15 cell line capable of stably expressing porcine Mx2 protein as well as preparation and application of the PK-15 cell line. The PK-15 cell line capable of stably expressing porcine Mx2 gene is constructed through a lentiviral vector system. A specific primer is designed according to the nucleotide sequence of the porcine Mx2 gene in a GeneBank database, recombinant lentivirus plasmids are constructed after target genes are amplified, HEK293T cell packaging lentivirus is transfected by utilizing a three-plasmid system, PK-15 cells are infected by the obtained recombinant lentivirus, and the PK-15 cell line for stably expressing the porcine Mx2 gene is obtained through puromycin screening. Experimental results show that the PK-15 cell line PK-15-poMx2 capable of stably expressing the porcine Mx2 protein is successfully constructed by the method disclosed by the invention. Compared with normal PK-15 cells, the expression quantity of Mx2 in the porcine Mx2 stable expression cell line is remarkably increased, and the porcine Mx2 stable expression cell line has an inhibiting effect on replication of PRV (Porcine Reproductive Virus). Therefore, the successful construction of the cell line for stably expressing the porcine Mx2 protein provides an important tool for deeply exploring the functional characteristics and the antiviral action mechanism of the porcine Mx2 protein.

The invention discloses a GPR21 expression vector and a construction method and application of a recombinant HEK293 cell based on the GPR21 expression vector, and belongs to the technical field of biology. PGMLV-CMV-HGPR21-EF1-ZsGreen1-T2A-Puro plasmids are constructed on the basis of a seamless cloning technology and are packaged into a lentivirus system for infecting HEK293 cells, puromycin is used for screening to obtain a genetic engineering cell line GPR21-HEK293 of stably transfected full-length GRP21, and the cell line is proved to be capable of improving the protein expression of the full-length GPR21 by 2.5 times, so that the gene engineering cell line GPR21-HEK293 can be used for efficiently expressing the full-length GPR21. The cell with high GPR21 expression provides a technical means for research on a ligand of a GRP21 orphan receptor and screening of drugs for treating inflammation, immunity, metabolism related diseases and the like by taking GRP21 as a target.

The application discloses an immortalized human hair papilla cell line and application thereof, and belongs to the technical field of immortalized cells, and particularly relates to a preparation method of an immortalized human hair papilla cell line; the method comprises the following steps: infecting human hair papilla cells by using an SV40 LT overexpression vector combined with a dyeing aid, treating the human hair papilla cells by using a puromycin screening solution, picking single clone cells by using a limited dilution method, and expanding the culture of the single clone cells to obtain the immortalized human hair papilla cell line; the dyeing aid comprises polybrene; compared with human hair papilla cells, the immortalized human hair papilla cell line prepared by the preparation method has higher cell activity and cell proliferation capacity, and can form 3D cell balls when cultured in vitro in a 3D mode, and the immortalized human hair papilla cell line retains the ability of inducing hair follicle formation, and can be applied to the fields of hair follicle tissue engineering, 3D cell culture or hair follicle organoid construction and the like.

The invention relates to the technical field of gene engineering technology, in particular to a preparation method of an efficient and stable gene knockout cell line. According to the AKAP11 gene knockout cell line provided by the invention, the 6th exon of the AKAP11 gene in the cell line is subjected to 3395bp frameshift deletion mutation, so that a coding frame is shifted, and protein translation is terminated in advance; after continuous subculture, the homozygous knockout phenotype is still maintained through PCR sequencing verification. A CRISPR / Cas9 system is combined with puromycin screening, the positive cloning rate reaches 30% or above, the construction period is shortened to 45 days, the constructed cell line can be directly used for AKAP11 related cell signal path research, disease model construction and targeted drug screening, and a reliable tool is provided for basic research and clinical transformation.

The invention discloses a method for knocking down or overexpressing a PDK4 gene of a BV2 microglial cell by utilizing a lentivirus technology and application of the method in ferroptosis research, and relates to the technical field of molecular biology and cytobiology, and the method comprises the following steps: designing an shRNA sequence through a full-length sequence of the PDK4 gene of a mouse so as to realize gene knocking down, or designing a cDNA sequence so as to carry out gene overexpression; the method comprises the following steps: constructing a lentiviral vector containing an shRNA sequence or a cDNA sequence, wherein the vector carries green fluorescent protein (GFP) and a purinase resistance gene; then carrying out lentivirus packaging and purification; the BV2 microglial cells are obtained and cultured; bV2 microglial cells are infected by lentivirus, and the BV2 microglial cell strain with stable knock-down or stable overexpression of the PDK4 gene is obtained through puromycin screening. According to the scheme, the cell strain capable of stably expressing PDK4 can be obtained, and the effect of the cell strain in ferroptosis is systematically researched.

There are described compositions comprising an effective amount of a combination of two or more components, said components selected from acacetin, ACTI peptide, alpha-lipolic acid, alprostadil, anisomycin, apigenin, ascorbic acid, astragalus, berberine, β-lapachone, β-hydroxy-beta-methyl-butyrate, Bacopa monnieri, catechin, catechol, chamomile, chrysin, coumestrol, curcumin, dinitrophenol, dinoprost, ellagic acid, (−)-epigallocatechin gallate, green tea extract, fisetin, genistein, ginsenoside RE, glabridin, 18-α-glycyrrhetinic acid, 18-β-glycyrrhetinic acid, glycyrrhizin, hydroquinone, isoquercitrin (EMIQ), kaempferol, kuromanin, leucine, lithium, luteolin, luteolin, luteolinidin, melatonin, menadione, 1-methylnicotinamide (MNA), methyl salicylate, myricetin, nadide, niacin (vitamin B3), nicotinamide (NAM), nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), nicotinic acid adenine dinucleotide (NaAD), nicotinic acid mononucleotide (NaMN), parsley (Petroselinium crispum), phenylephrine, pokeweed mitogen, 15-Δ prostaglandin J2, puromycin, quercetin, quinolinic acid, retinoic acid, trichostatin A, troxrutin, rutin, tryptophan, vitamin D3, withaferin A, wortmannin and zinc (including salts thereof).

The invention belongs to the technical field of biology, and particularly discloses a cell line capable of stably expressing an nsP4 gene and application of the cell line in preparation of a replication-deficient chikungunya virus. The inventor uses retrovirus for expressing CHIKV nsP4 to infect hamster kidney cells (hamster kidney cells), after puromycin screening, verification of passage stability of monoclonal cells, protein expression stability, replication-deficient virus titer and the like is carried out, finally, a cell line which can be used for continuous passage of CHIKV replication-deficient viruses and has high titer is obtained, and the preservation number is CCTCC NO: C2025281. The replication-deficient virus CHIKV-delta nsP4 generated by the RNA of the nsP4 gene deleted from the trans-complementary CHIKV of the cell line has good safety, immunogenicity and immune protection efficacy, can effectively resist the challenge infection of wild viruses, and can be used for preparing a safe and effective CHIKV novel vaccine candidate strain.

The invention discloses a stably transfected cell strain for detecting activity of Wnt and / or R-spondin family cytokines as well as a construction method and application of the stably transfected cell strain, and belongs to the technical field of biological medicines. Comprising the following steps: preparing target plasmids: respectively connecting a SuperTOP sequence and a SuperFOP sequence to two different expression plasmids, and respectively connecting luciferase reporter genes to obtain a target plasmid A and a target plasmid B; respectively transfecting the target plasmid A and the target plasmid B into human kidney epithelial cells, culturing in a puromycin-containing culture medium, and sorting positive cells according to labeled fluorescence GFP (Green Fluorescent Protein) to obtain stably transfected cells A and stably transfected cells B. The method has the advantages that activity detection of Wnt family and R-spondin family cell factors is carried out on stably transfected cells, operation is simple, the detection period is shortened by 25%, sensitivity is improved by at least 50%, and the method has high application prospects and economic value.

The invention provides a HaCaT cell model capable of stably expressing green fluorescent protein as well as a construction method and application of the HaCaT cell model, and belongs to the technical field of cell model construction. The construction method of the cell model comprises the following steps: mixing a PiggyBac-TetOn-CMV-P2A-EGFP-Puro plasmid, a Super PiggyBac Transspobase plasmid, a cell culture solution and a transfection reagent, so as to obtain a GFP (Green Fluorescent Protein) transfection reagent; mixing HaCaT cells with the obtained GFP transfection reagent, transfecting, adding puromycin for screening after the transfection is finished, and stopping adding puromycin after the screening is finished, so as to obtain the cells which are the HaCaT cell model for stably expressing the green fluorescent protein. The cell model can be used for revealing the interaction between skin cells, screening drugs for preventing or treating skin diseases and evaluating the efficacy of cosmetics.

The invention discloses a bispecific antibody nanodisk as well as a preparation method and application thereof. The preparation method provided by the invention comprises the following steps: connecting a CD3 antibody gene and a PD-L1 antibody gene through a hydrophobic transmembrane region, cloning to a pCDH vector, packaging a lentivirus infected 293T cell, and screening through puromycin to obtain the 293T cell for stably expressing the CD3 * PD-L1 bispecific antibody; carrying out ultrasonication and gradient centrifugation to collect CD3 * PD-L1 bispecific antibody cell membrane vesicles; and mixing 22A polypeptide, the vesicles and DMPC according to a mass ratio of 1: 1: 5, rotating at a low speed of 3-5 DEG C, and uniformly mixing for 11-13 hours to obtain the bispecific antibody nanodisk. According to the scheme, the tumor targeting effect of the bispecific antibody can be improved, and the anti-tumor effect is enhanced.

The invention discloses an immortalized egg shell gland epithelial cell line and application thereof. The invention belongs to the technical field of biology, and particularly relates to an immortalized egg shell gland epithelial cell line and application thereof. The construction method of the immortalized egg shell gland epithelial cell line comprises the following steps: 1) obtaining SV40 overexpression lentivirus; 2) transfecting the lentivirus obtained in the step 1) to chicken primary eggshell gland epithelial cells; amplifying the transfected cells for 3-4 generations, and screening puromycin to obtain successfully transfected eggshell gland epithelial cells; and (3) carrying out subculture on the successfully transfected eggshell gland epithelial cells obtained in the step (2) to obtain the stably subcultured immortalized eggshell gland epithelial cells. The immortalized egg shell gland epithelial cell provided by the invention can provide a reliable cell model for researching related functions of poultry egg shell glands, and has an important value for developing a novel feed additive capable of improving chicken genital tract health and egg shell quality.

The invention relates to a joint and application thereof. A linker according to the present invention comprises: a binding part having a structure capable of binding to a desired genetic information substance; and at least two or more puromycin-like substances capable of covalently binding to the C-terminal of a desired peptide.

The invention discloses a BHK-21 cell line capable of stably expressing a human ENT1 gene and a construction method and application thereof, the cell line is hamster kidney tissue fibroblast BHK-ENT1, the cell line carries the human ENT1 gene, and the ENT1 provides substances and energy basis necessary for replication for viruses by maintaining the steady state of nucleotide and energy in the cell, so that the human ENT1 gene can stably express the human ENT1 gene. The virus titer generated after the Newcastle disease virus infects host cells is improved. The construction method of the cell line comprises the following steps: amplifying a human ENT1 gene and constructing a stable expression vector, co-transfecting the stable expression vector and packaging plasmids psPAX2 and pMD2. G to an HEK-293T cell, culturing, and collecting a supernatant rich in lentiviral particles; and infecting the BHK-21 cell with the collected lentivirus, screening through a puromycin drug, and carrying out enlarged culture, so as to obtain the BHK-ENT1 cell.

The invention discloses a construction method of an immortalized chicken follicle granule cell line. The method specifically comprises the following steps: constructing an SV40LT gene overexpression lentiviral vector system, packaging and preparing a high-titer lentivirus; after chicken follicular granular cells are separated, lentivirus is used for transduction and puromycin resistance screening, and finally, an immortalized cell line is obtained through subculture. The constructed immortalized cell line has the following characteristics: continuous and stable passage can be realized, the biological functions of normal granular cells are reserved, and marker protein FSHR is specifically expressed; compared with primary cells, the proliferation capacity is remarkably enhanced, and the cell aging level is remarkably reduced. The method disclosed by the invention is simple and convenient to operate and high in efficiency, provides a reliable in-vitro model for research on follicular development, ovulation mechanism, reproductive endocrine regulation and the like of chicken, has important theoretical research and application values, and meanwhile, has a wide application prospect in reproductive development research related to reproduction in the poultry industry.

The invention discloses a reversible gene silencing stable cell line construction method based on inducible shRNA (short hairpin Ribonucleic Acid) expression, which belongs to the technical field of biology and comprises the following steps: (1) transferring a vector containing a drug inducible promoter system and a shRNA sequence of a targeted specific gene into a target cell through lentiviral transfection, screening through puromycin to obtain a monoclonal cell population of a stable integration vector; (2) adding doxycycline into the culture medium of the monoclonal cell population, and inducing shRNA expression to silence target protein; and (3) verifying the expression recovery of the target protein by removing doxycycline, and confirming the reversibility of the system. According to the invention, shRNA expression is controlled through the drug-induced promoter system, reversible silencing of specific genes is realized, and the drug-induced promoter system has the characteristics of high induction efficiency, low background expression and good stability.

This invention discloses a stable A549 expression cell line constructed based on a dCas9-VP64 transcriptional activation system and its applications. Based on the dCas9-VP64 transcriptional activation system, this invention yielded a stable A549 expression cell line with the highest relative expression level of dCas9 protein after puromycin selection. Subsequently, by introducing sgRNAs targeting one or more genes into this cell line, the expression of endogenous genes in lung cancer cells can be activated, and related functional gene studies can be conducted. Furthermore, this invention prepares an A549 cell line with significantly reduced proliferation rate by introducing sgRNA targeting the TP53 binding site in the LTR5Hs sequence into this cell line. This invention is the first to discover a novel target for lung cancer treatment, which is of great significance for the treatment and mechanistic research of lung cancer.

The invention discloses an immortalized human hair papilla cell line and application thereof, belongs to the technical field of immortalized cells, and particularly relates to a preparation method of the immortalized human hair papilla cell line. The method comprises the following steps: infecting human hair papilla cells by adopting an SV40LT overexpression vector combined with a dyeing assistant, treating the human hair papilla cells by using a puromycin screening solution, selecting monoclonal cells by using a limited dilution method, and performing amplification culture to obtain an immortalized human hair papilla cell line; the dyeing assistant comprises polybrene; compared with human hair papilla cells, the immortalized human hair papilla cell line prepared by the preparation method has higher cell activity and cell proliferation capacity, forms 3D cell balls during in-vitro 3D culture, retains the ability of inducing hair follicle formation, and can be applied to the fields of hair follicle tissue engineering, 3D cell culture or hair follicle organ construction and the like.