CN104388435A - Construction and application of goat adiponectin gene eukaryotic expression vector - Google Patents
Construction and application of goat adiponectin gene eukaryotic expression vector Download PDFInfo
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- CN104388435A CN104388435A CN201410581934.9A CN201410581934A CN104388435A CN 104388435 A CN104388435 A CN 104388435A CN 201410581934 A CN201410581934 A CN 201410581934A CN 104388435 A CN104388435 A CN 104388435A
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to construction and application of a goat adiponectin gene eukaryotic expression vector. The nucleotide sequence of a goat adiponectin gene coding region is shown in the sequence table SEQ ID No.1. A complete coding region fragment of a goat adiponectin gene is subjected to double digestion and then is connected to a pEGFP-N1 vector subjected to double digestion, so that the goat adiponectin protein eukaryotic expression vector can be constructed. The eukaryotic expression vector construction method is simple and feasible, the expression of goat adiponectin protein is enhanced efficiently, green fluorescent protein is included, and expression and positioning of the goat adiponectin protein eukaryotic cell can be detected. The eukaryotic expression vector provided by the invention can also be used for researching the function of adiponectin protein in a goat fat deposition process.
Description
Technical field
The invention belongs to technical field of bioengineering, be specifically related to a kind of construction process of goat apm 1 gene carrier for expression of eukaryon and the application in goat fatty deposits thereof.
Background technology
Animal body fat tissue is important energy storage organ, is again important endocrine organ.Fatty tissue can secrete multiple Adipocyte Factor, as adiponectin (Adiponectin, AdipoQ), tumour necrosis factor (Tumor necrosis factor α, TNF α), leptin (Leptin, LEP), interleukin-6 (Interleukin-6, IL-6), phylaxin (Resistin, RETN) etc.AdipoQ has very high concentration (5 ~ 30 μ g/mL) in blood, reaches 0.01% ~ 0.05% of Total plasma protein.AdipoQ plays an important role in many middle physiological functions, can strengthen the insulin sensitivity of type ii diabetes patient, be used for the treatment of type ii diabetes; Regulation and control glycolipid metabolism, suppresses the hydrolysis of glycogen, promotes the oxidation of blood free fatty acid and the absorption of glucose sugar; Suppress the phagocytic activity of granulocyte, mononuclear macrophage Colony forming and scavenger cell, play anti-inflammatory effect; Suppress vascellum tunica interna incrassation and smooth muscle cell proliferation, play atheroma effect etc.AdipoQ arrives each organ of whole body by blood circulation, in different forms with corresponding receptors bind, all plays an important role in whole body.
Research finds in animal imitating test, change the content of adiponectin or can regulate and control the metabolism of glucose and lipid acid with restructuring adiponectin ex vivo treatment of cells and muscle, it is by promoting glucose transporter 4(GLUT4) absorption increasing glucose is shifted to cytolemma, activate absorption and oxidation that Adenylate cyclase (AMPK), p38 mitogen activated protein kinase (p38-MAPK) and Peroxisome Proliferator-activated Receptors (PPAR α) path increase lipid acid, but the AdipoR1/2 level that in this, effect can be caused by Leptin reduces and weakens
[1].AdipoQ can improve the insulin resistant that high sugar brings out, and the triglyceride level (TG) reduced in serum and free fatty acids (FFA) level, suppress lipoproteinesterase glucose that is active and Regular Insulin regulation and control to produce
[2], find that the expression amount of AdipoR1 and AdipoR2 and insulin sensitivity are proportionate in addition
[3].In subjects with hyperglycemia skeletal muscle, the expression amount of AdipoR1 reduces by 50%, thus the glucose transporter 4(GLUT4 that inhibit gAd to regulate and control) transposition, reduce the absorption to glucose sugar, lipid acid and oxidation; But enhance the glucose-lipid metabolism function of full-length AdipoQ
[4].
Wu etc.
[5]utilize dexamethasone, isobutyl methylxanthine and insulin combination inductor process sheep SMSCs, result shows that SMSCs can deposit fat and be divided into class adipocyte.Teboul etc.
[6]research finds that lipid acid can make C2C12 cell deposition fat and be divided into class adipocyte, detects that the expression amount of related fatty acids synthetic gene significantly raises simultaneously.Research also find high concentration glucose by
sREBP-1cthe de novo synthesis of lipid acid in path regulation and control myotube, and all detect that the fatty acid synthesis gene expression amounts such as SREBP-1c, ACC and FAS have significance to raise and are divided into adipocyte etc. at mRNA and protein level, show that SMSCs is the Muscle-derived Stem Cells with many differentiation potentials
[7].AdipoQ is as a kind of important Adipocyte Factor, and the fermentation such as the glycolipid metabolism adjustment in muscle play an important role.Therefore can be regulated and controled the synthesis of lipid acid in muscle by AdipoQ, increase intramuscular fat content, thus improve goat meat quality.
[1] Yamauchi T, Kamon J, Ito Y, et al. Cloning of adiponectin receptors that mediate antidiabetic metabolic effects[J]. Nature, 2003, 423(6941): 762-769.
[2] Combs TP, Pajvani UB, Berg AH, et al. A transgenic mouse with a deletion in the collagenous domain of adiponectin displays elevated circulating adiponectin and improved insulin sensitivity[J]. Endocrinology, 2004, 145(1): 367-383.
[3] Civitarese AE, Ukropcova B, Carling S, et al. Role of adiponectin in human skeletal muscle bioenergetics[J]. Cell metabolism, 2006, 4(1): 75-87.
[4] Fang X, Palanivel R, Zhou X, et al. Hyperglycemia- and hyperinsulinemia-induced alteration of adiponectin receptor expression and adiponectin effects in L6 myoblasts[J]. J Mol Endocrinol, 2005, 35(3): 465-476.
[5] Wu H, Ren Y, Li S, et al. In vitro culture and induced differentiation of sheep skeletal muscle satellite cells[J]. Cell Biol Int, 2012, 36(6): 579-587.
[6] Teboul L, Gaillard D, Staccini L, et al. Thiazolidinediones and fatty acids convert myogenic cells into adipose-like cells[J]. J Biol Chem, 1995, 270(47): 28183-28187.
[7] Guillet-Deniau I, Pichard A-L, Koné A, et al. Glucose induces de novo lipogenesis in rat muscle satellite cells through a sterol-regulatory-element-binding-protein-1c-dependent pathway[J]. J Cell Sci, 2004, 117(10): 1937-1944。
Summary of the invention
The object of the present invention is to provide a kind of construction process of goat apm 1 gene carrier for expression of eukaryon and the application in goat fatty deposits thereof.
the present invention is realized by following technology:according to the multiple clone site of pEGFP-N1 carrier and the coding region sequence of goat apm 1 gene; design with protectiveness base, end respectively with the primer of Hind III and BamH I restriction enzyme digestion sites, amplified production comprises the coding region sequence of goat apm 1 gene.Be built between the Hind III of pEGFP-N1 carrier and BamH I two restriction enzyme sites.Obtain goat apm 1 gene different green fluorescent protein fusion expression vector pEGFP-N1-AD.
effect of the present invention is:utilizing pair of primers to carry out pcr amplification can the complete coding region of cloned goat apm 1 gene, this construction of eukaryotic expression vector method simple possible, the expression of goat tallow connection fibroin can be strengthened efficiently, and all comprising green fluorescent protein, is the important technology of research goat tallow connection fibroin function.
More detailed technical scheme refers to the embodiment in " embodiment ".
Accompanying drawing explanation
Sequence table SEQ ID NO:1 is the nucleotide sequence of the goat apm 1 gene that the present invention clones.
Fig. 1 is the structure iron of the carrier for expression of eukaryon pEGFP-N1-AD that the green fluorescent protein of goat apm 1 gene merges.
Fig. 2 be after the green fluorescent protein fusion expression vector pEGFP-N1-AD of goat tallow connection fibroin imports goat skeletal muscle satellite cell by transient transfection lipid acid synthesis and become fat differentiation associated gene (
adipoR2,
aCC,
fAS,
sREBP-1, AdipoR1,
c/EBP αwith
pPAR γ) change of expression amount.
Embodiment:
embodiment 1:(mono-) clone of goat apm 1 gene coding region sequence
1. the extraction of total serum IgE
(1) appropriate liquid nitrogen is poured in mortar precooling, after temperature reduces, tissue is put into mortar, add suitable liquid nitrogen again and will sample be organized to be ground into powder, then powdered sample 100 mg is got in the EP pipe of 1.5 mL, add the Trizol reagent of 1 mL precooling, concussion mixing, room temperature leaves standstill 5 min.
(2) add chloroform 200 μ L, 15 sec mixings that vortex instrument vibrates, room temperature leaves standstill 5 min, then 12,000 × g centrifugal 15 min under 4 DEG C of conditions.
(3) the 500 μ L that makeed an appointment by upper water are transferred to the EP pipe of another new 1.5 mL, add isopyknic pre-cold isopropanol, put upside down mixing for several times gently, are placed in room temperature and leave standstill 10 min, then 12,000 × g centrifugal 10 min under 4 DEG C of conditions.
(4) remove supernatant, add 75 % DEPC alcohol 1 mL of 4 DEG C of precoolings, leave standstill 5 min, then 7,500 × g centrifugal 5 min under 4 DEG C of conditions on ice;
(5) remove supernatant, draw unnecessary liquid with liquid-transfering gun, drying at room temperature precipitates.
(6) add appropriate DEPC water dissolution RNA according to the amount of the precipitation of RNA, and be stored in-80 DEG C of Ultralow Temperature Freezers.
2.
the synthesis of cDNA
Reverse Transcription box PrimeScript RT reagent Kit is utilized to carry out the synthesis of cDNA Article 1 chain.The first step removing genomic dna: the RNA 1 μ g getting said extracted, 5 × gDNA Eraser Buffer 2 μ L, gDNA Eraser 1 μ L, adds Rnase Free dH
2o to 10 μ L, brief centrifugation after mixing, reacts 2 min under 42 DEG C of conditions gently.Second step is the synthesis of cDNA: in substance system, add 5 × PrimerScript Buffer 2 reagent 4 μ L, PrimeScript RT Enzyme Mix I 1 μ L, RT Primer Mix 1 μ L, mends RNase Free dH
2o to 20 μ L, brief centrifugation after mixing, reacts 15 min under 37 DEG C of conditions, is then warming up to 85 DEG C of reaction 5 sec deactivation synthetic enzyme.The cDNA of synthesis is in-20 DEG C of preservations.
3.
rT-PCR amplification goat apm 1 gene
The cDNA that is derived from fatty tissue is utilized to carry out the clone of genes involved for template.Reaction system is: cDNA template 2 μ L, each 0.8 μ L(20 pM of upstream and downstream primer), 2 × Master Mix Taq enzyme 10 μ L, adds sterilizing ddH
2o to 20 μ L.
4.
the clone of apm 1 gene
(1) according to PMD 19-T carrier operation instruction, add 0.5 μ L PMD 19-T Vector to 1.5 mL centrifuge tubes, the PCR of 4.5 μ L reclaims product, and 5 μ L Solution I, mix gently, and is placed in 16 DEG C of Water Under bath connection 5 h.
(2) competent cell is taken out to be placed on from Ultralow Temperature Freezer slowly melt on ice, draw 30 μ L competent cells and mix with above-mentioned connecting fluid, mix gently, then ice bath 30 min.
(3) 42 DEG C of heat-shock transformed 45 sec of Water Under bath, then place 1 ~ 2 min on ice.
(4) what in above-mentioned mixed solution, add 37 DEG C of preheatings does not contain antibiotic LB liquid nutrient medium 800 μ L, is placed in upper cultivation 45 min of 37 DEG C of constant-temperature tables (200 r/min), recovery competent cell.
(5) 4,000 × g centrifugal 10 min under room temperature, remove 650 μ L supernatants, are blown and beaten by remaining substratum and mix, be coated on uniformly containing on antibiotic LB solid medium flat board with precipitation.First being just placed in 37 DEG C of incubators cultivates 1 h, and bacterium liquid to be mixed is inverted cultivation 12 ~ 16 h after being absorbed by solid medium.
the qualification of apm 1 gene and order-checking
After bacterium colony is formed, select single bacterium colony, be inoculated in containing in microbiotic LB liquid nutrient medium, be placed in upper cultivation 6 ~ 8 h of 37 DEG C of constant-temperature tables (200 r/min), to be observed in substratum, form cloud after can carry out the detection of bacterium liquid.Mixed by bacterium liquid, draw bacterium liquid 1 μ L as DNA profiling, performing PCR of going forward side by side increases, band for the purpose of whether 1.5% agarose detects.After detection, the bacterium liquid containing object band is delivered to the order-checking of Shanghai Sheng Gong Bioisystech Co., Ltd.
embodiment 2:(mono-) structure of goat tallow connection fibroin carrier for expression of eukaryon
1. vector construction
(1) Primer Premier 5.0 software analysis goat apm 1 gene CDS region sequence is utilized, find and can not cut the restriction enzyme in this gene C DS district by enzyme, and contained by pEGFP-N1 carrier restriction enzyme site information determination restriction enzyme Hind III and BamH I.According to restriction enzyme site sequence and CDS region sequence information design primer amplification goat apm 1 gene CDS district, forward primer (F1): 5 ' (5 '-CCAAGCTTATGCTGCTGCTGGGAGCTCT-3 ') and reverse primer (R1): 3 ' (5 '-CGGGATCCCATTCGATGTTATGGTAGAGAA-3 ').
(2) according to operation instructions, enzyme cuts pEGFP-N1 carrier and goat apm 1 gene PCR primer.Glue reclaims digestion products, utilizes T4 DNA ligase to spend the night connection.
(3) by vector in competent cell.
(4) select single bacterium colony, liquid nutrient medium expanding propagation, PCR detects containing after goal gene band, bacterium liquid is delivered to the order-checking of Shanghai Sheng Gong Bioisystech Co., Ltd.
Utilize the primer containing restriction enzyme site to carry out pcr amplification goat apm 1 gene CDS sequence, glue reclaims PCR primer.Restriction enzyme Hind III and BamH I enzyme cut PCR primer and pEGFP-N1 carrier for expression of eukaryon, enzyme is cut rear gel electrophoresis and is separated rear glue recovery product, then by T4 ligase enzyme, goat apm 1 gene CDS district is connected into pEGFP-N1 carrier, proceed in competent cell and expand numerous rear order-checking, obtain correct recombinant vectors, carrier lengths is 5420 bp, and structure is as Fig. 1.
embodiment 3:(mono-) apm 1 gene is to the effect of goat fatty deposits
1. cell cultures and transient transfection
(1) SMSCs is with 5 × 10
4/ mL density is inoculated in six orifice plates, and growth medium is cultivated, and is placed in 37 DEG C, 5% CO
2cultivate under condition.
(2) until cell proliferation to when to stick bottom plate about 80%, substratum is replaced by antibiotic-free and cultivates based 1.5 mL.
(3) illustrate according to transfection assay, 5 min in the low serum of 250 μ L, antibiotic-free substratum by 10 μ L liposomes and 4 μ g plasmid incubated at room respectively, then mix the two, incubated at room 20 min, join in above cell culture medium, rock mixing substratum gently.
(4) remove former substratum after transfection 6 h, utilize PBS rinsing cell 3 times, change fresh growth medium, continue cultivation 24 ~ 36 h.
(5) fluorescent microscope is utilized to detect expression amount and the Subcellular Localization of Transfected cells Green fluorescin (GFP)
Result shows: carry out transient transfection when SMSCs to be cultured at the bottom of confluent culture ware 80% ~ 90%, after transfection 24 h, fluorescent microscope detects green fluorescence, shows that recombinant vectors is successfully expressed in SMSCs.AdipoQ transfection group compares discovery with pEGFP-N1 blank group fluorescence, and AdipoQ transfection group green fluorescence is only expressed in tenuigenin, does not detect in nucleus.
2.
adipoQ is to the expression regulation of Genes Associated with Lipid Metabolism
Collect control group, each 3 holes of transfection group cell, extract total serum IgE, detect qualified rear reverse transcription synthesis cDNA.QPCR is utilized to detect
adipoR1,
adipoR2synthesize with lipid acid and become fat differentiation associated gene
aCC,
fAS,
c/EBP α,
sREBP-1with
pPAR γrelative expression quantity, utilize 2 equally
-Δ Δ Ctthe relative expression quantity of each gene of methods analyst.
In qPCR detection SMSCs, after process LAN AdipoQ gene, lipid acid synthesis and one-tenth fat differentiation associated gene expression amount have significance to change (test sample repeat number is 3), AdipoR2, ACC, FAS and SREBP-1 gene expression amount significantly raises, raise 1.85,4.55,4.88 and 4.25 times respectively, as Fig. 2; And the expression amount of AdipoR1, C/EBP α and PPAR γ is without considerable change, show that AdipoQ gene can raise the expression of AdipoR2, ACC, FAS and SREBP-1 gene, as Fig. 2.
SEQUENCE LISTING
<110> Sichuan Agricultural University
The structure of <120> goat apm 1 gene carrier for expression of eukaryon and application
<130> 2014
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 720
<212> DNA
<213> goat (Capra hircus)
<400> 1
atgctgctgc tgggagctct tctactgctg ctagccctgc ccagtcatgg ccaggacacc 60
atgcaagggc ccccgctgcc caagggggcc tgcacaggtt ggatggcagg cattccaggg 120
catcctggcc acaacgggac cccaggccgg gatggcagag atggcacccc tggtgagaag 180
ggtgagaaag gagatccagg tcttgttggt cctaagggtg acaccggtga aactggaatc 240
actgggattg aaggtccccg aggctttcca ggagtcccag gcagaaaggg agaacctgga 300
gaaagtgcct atgtataccg ctcagcattc agtgtgggac tggagagccg ggtcactgtc 360
cccaatgttc ccattcgctt taccaagatc ttctacaatc agcaaaacca ctatgatggc 420
accactggca aattcctctg caatattccc gggctgtact acttctccta ccacattact 480
gtctacttga aggatgtgaa ggtcagcctc tacaagaacg acaaggccct gctcttcacc 540
cacgaccagt tccaggacca gaacgtggac caggcctctg gctccgtgct cctctatctg 600
gagaagggtg accaagtctg gctccaggtg tacgagggtg aaaatcacaa tggggtctat 660
gcggataacg tccatgactc caccttcaca ggcttccttc tctaccataa catcgaatga 720
Claims (4)
1. a method for cloned goat apm 1 gene coding region sequence, its nucleotide sequence is as described in sequence table SEQ ID NO:1.
2. goat apm 1 gene eukaryon recombinant plasmid; it is characterized in that being formed by the nucleotide sequence of the goat apm 1 gene in sequence table described in SEQ ID NO:1 and pEGFP-N1 vector construction; wherein utilize and carry out pcr amplification with the primer of Hind III and BamH I restriction enzyme digestion sites respectively with protectiveness base, end, the forward and reverse primers DNA sequences of PCR reaction is as follows:
Forward primer (F1): 5-CCAAGCTTATGCTGCTGCTGGGAGCTCT-3;
Reverse primer (R1): 5-CGGGATCCCATTCGATGTTATGGTAGAGAA-3.
3. prepare the method for eukaryon recombinant plasmid as claimed in claim 2, according to following steps:
(1) with the cDNA of goat fatty tissue for template, the coding region of pcr amplification goat apm 1 gene;
(2) respectively double digestion is carried out to the coding region of the goat apm 1 gene after purifying and pEGFP-N1 plasmid with Hind III and BamH I restriction enzyme, carry out enzyme with ligase enzyme T4 DNA Ligase to connect, then product conversion bacillus coli DH 5 alpha will be connected, coating is added with the antibiotic flat board of Kan, screening positive clone.
4. according to the goat apm 1 gene carrier for expression of eukaryon that method described in right 3 obtains, the application acted in goat tallow connection fibroin Subcellular Localization and research goat fat deposition process.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962629A (en) * | 2015-06-29 | 2015-10-07 | 中国农业科学院兰州畜牧与兽药研究所 | Specific primer and fluorescent quantitative detection kit detecting cattle ADIPOQ gene mRNA expression level |
| CN105823888A (en) * | 2016-04-08 | 2016-08-03 | 福建农林大学 | Subcellular localization kit constructed through sugarcane streak mosaic virus P3N-PIPO |
| CN114317417A (en) * | 2020-10-10 | 2022-04-12 | 重庆市畜牧科学院 | Method for inducing skeletal muscle satellite cell to generate lipid droplets |
-
2014
- 2014-10-28 CN CN201410581934.9A patent/CN104388435A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| NCBI: "Capra hircus adiponectin,C1Q and collagen domain containing(ADIPOQ),mRNA;NM_001287573.1", 《NCBI》 * |
| 周静等: "人脂联素真核表达载体的构建及其表达", 《第三军医大学学报》 * |
| 潘逸茹等: "人脂联素重组克隆载体构建", 《现代生物医学进展》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962629A (en) * | 2015-06-29 | 2015-10-07 | 中国农业科学院兰州畜牧与兽药研究所 | Specific primer and fluorescent quantitative detection kit detecting cattle ADIPOQ gene mRNA expression level |
| CN105823888A (en) * | 2016-04-08 | 2016-08-03 | 福建农林大学 | Subcellular localization kit constructed through sugarcane streak mosaic virus P3N-PIPO |
| CN114317417A (en) * | 2020-10-10 | 2022-04-12 | 重庆市畜牧科学院 | Method for inducing skeletal muscle satellite cell to generate lipid droplets |
| CN114317417B (en) * | 2020-10-10 | 2023-05-12 | 重庆市畜牧科学院 | Method for inducing skeletal muscle satellite cells to generate lipid droplets |
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