30 results about "Neomycin Resistance Gene" patented technology

The invention discloses a salivary gland tissue specific transgenic carrier and a construction method thereof. The salivary gland tissue specific transgenic carrier is obtained by emerging an upstream regulation region sequence of a mice parotid gland protein promoter and a visual screened neo-EGFP combined double-label to a piggyBac transposon system and a Lox-Cre enzyme system; the transgenic carrier can make protein express in a particular tissue to achieve a high-efficient large-section emerging efficiency. The carrier also has a green luminescence label which is convenient for screening and a neomycin resistance gene which is convenient for eukaryotic cell screening, and makes the screening of transgenic cells and identification of transgenic animals be simpler, more direct, and more accurate. The transgenic carrier provided by the invention is a salivary gland specific expression high-efficient integration universal carrier, and is capable of being applied to fields of transgenic animals such as transgenic mice and pig, and the like. The invention clones the upstream regulation region of FVB mice for the first time, the cloning method is simple and high efficient, and the difficulty in construction of a large section carrier is effectively overcome.

The invention discloses a promoter trapping type targeting vector PIII-MSTN for knockout of a bovine myostatin MSTN gene. The vector can knock all of a first exon and a second exon and most of a third exon of the bovine MSTN gene; a knockout nucleotide sequence is shown as a SEQ ID NO:1; a PIII MSTN nucleotide sequence is shown as a SEQ ID NO:3, and contains homologous arm sequences about 1.3kb long and 6.8kb long of the bovine MSTN gene, a green fluorescent protein gene EGFP without promoter and a neomycin resistance gene Neor expression framework with promoter PGK. The promoter trapping type targeting vector PIII-MSTN provided by the invention can be used for the cultivation of transgenic beef cattle with MSTN gene knocked out.

The present invention is to provide TLR1 knockout mice specifically recognizing mycobacterial lipoproteins / lipopeptides, being useful to clarify the role of TLR1 in vivo, or a method for screening substances promoting or suppressing the response to mycobacterial lipoproteins / lipopeptides by using the same. TLR1 genes are separated from murine genomic library, the genomic part containing the intracellular and transmembrane domain of the TLR1 gene is replaced with a neomycin resistant gene, HSV-tk gene being gene encoding thymidine kinase into 3′ end is introduced, ES cell clones having double resistance to G418 and Gancyclovir are screened, and ES cell clones are injected into the blastocyst of C57BL / 6 mice, to generate TLR1 knockout mice through the germline.

The invention relates to a promoter replacement method for improving the output of the fecula liquefied bacillus fengycin, belonging to the field of biotechnology. The method includes: using a PCR method to amplify and obtain a 450bp homologous sequence from fecula liquefied bacillus ES-2 strain genome, amplifying a promoter P59 sequence from a plasmid pHB201, connecting together the two DNA fragments, and cloning into a plasmid pMD19-T, and connecting a neomycin resistance gene as a selection marker, thereby constructing a homologous integration vector; and then transforming the constructed homologous integration vector into the wild fecula liquified bacillus ES-2, wherein, the fengycin output of the promoter strain is 5704.77 +- 258.89mg / L, which improves about 0.65 times than the fengycin output of the wild bacteria.

The present invention provides a method for preparing human-derived feeder cells that can support the growth of human-derived feeder cells that can support the growth of human embryonic stem cells, comprising the following steps: S1, placing human bone marrow mesenchymal stem cells in a culture medium for subculture ; S2, infecting the human bone marrow mesenchymal stem cells subcultured in S1 with retroviral particles containing the hygromycin resistance gene to obtain human bone marrow mesenchymal stem cells; S3, infecting the human bone marrow mesenchymal stem cells containing the Wnt3a gene and the neomycin resistance gene The retroviral particles infected human bone marrow mesenchymal stem cells to obtain TW2R cells; S4, the lentiviral particles containing E-cadherin gene and puromycin resistance gene were transfected into TW2R cells to obtain TWE3R cells. The human feeder layer cells prepared by the present invention can stably express the E-cadherin protein, and the stable expression of the E-cadherin gene will not affect the proliferation ability of the TWE3R cells.

The invention discloses a ribonucleic acid (RNA) interference vector for bovine genome locus specificity recombination, a vector construction method and application and belongs to the field of biotechnology. The interference vector comprises the following sequentially connected parts: an HSV-TK expression element, A digestion locus for vector linearization, a homologous recombination arm 1 in homologous recombination with a bovine scaffold sequence upstream, a human U6 promoter sequence, a polyclonal digestion locus, a human U6 stop subsequence, a neomycin resistance gene containing a PCMV promoter, a homologous recombination arm 2 in homologous recombination with a bovine scaffold sequence downstream, a replication origin gene and an ampicillin resistance screening gene. The RNA interference vector achieves accurate target shooting on bovine derived cell bovine, and brings convenience to screening, insertion locus and insertion element do not affect the integral structure of chromosome and cause no non-research gene expression effect on genome, and RNA interference vector provides convenience for research.

The invention discloses a cell line for stable expression of a chicken interleukin 10 protein, as well as a construction method and application thereof. The construction method disclosed by the invention comprises the steps of cloning a chicken interleukin 10 encoding gene after removal of a signal peptide into a eukaryotic expression vector containing a neomycin resistance gene in a fixed direction to obtain a recombinant plasmid, transfecting cells of a Chinese hamster ovary cell line CHO-K1, and performing G418 pressurized screening and purification to obtain a cell line CHO-chIL-10M for stable and high-efficient expression of a chIL-10 protein, wherein the microorganism collection number is CGMCC NO. 7659. The cell line CHO-chIL-10M constructed by the construction method disclosed by the invention has extensive application prospects in preparation of recombinant chicken interleukin 10 proteins and chicken interleukin 10 monoclonal antibodies and other aspects.

The invention discloses a RNA interference vector based on site-specific recombination, and construction method and application of the same, belonging to the field of biotechnology. The interference vector provided by the invention sequentially comprises the following connected parts: a homologous recombination arm 1 in homologous recombination with the upstream of a certain intron region of the integrin receptor beta 3 gene of pig, a human U6 promoter sequence, a multiple cloning enzyme digestion site, a human U6 terminator sequence, a new Neomycin resistant gene containing a PCMV (Porcine cytomegalovirus) promoter, a homologous recombination arm 2 in homologous recombination with the downstream of a certain intron region of the integrin receptor beta 3 gene of pig, a replication origin gene, an ampicillin resistant screened gene and a enzyme digestion site for vector linearization. The interference vector provided by the invention realizes accurate targeting to swine-origin cell genome, and facilitates screening, meanwhile, the influence of insertion of segment on the gene expression can be predicted, which is convenient for the following experiment.

An expression carrier for effectively screening target protein is prepared through introducing an internal ribosome to the eukaryotic expression carrier containing CMV promoter and dhfr amplification system, linking the fusion gene of anti-erbB2-seFv-Fc gene and IL-2 to the neomycin-resistant gene, site mutation to Neo gene by PCR, and configuring the expression carrier pCMV-HFI-mNeo.