CN103992409B - Fusion protein and its application for rejecting marker gene - Google Patents
Fusion protein and its application for rejecting marker gene Download PDFInfo
- Publication number
- CN103992409B CN103992409B CN201410158270.5A CN201410158270A CN103992409B CN 103992409 B CN103992409 B CN 103992409B CN 201410158270 A CN201410158270 A CN 201410158270A CN 103992409 B CN103992409 B CN 103992409B
- Authority
- CN
- China
- Prior art keywords
- cpp5
- cre
- cell
- pig
- rejected
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000003550 marker Substances 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 27
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 23
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 23
- 108090000790 Enzymes Proteins 0.000 claims abstract description 13
- 102000004190 Enzymes Human genes 0.000 claims abstract description 13
- 108700019146 Transgenes Proteins 0.000 claims abstract description 12
- 210000002950 fibroblast Anatomy 0.000 claims abstract description 8
- 210000001082 somatic cell Anatomy 0.000 claims abstract description 8
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 claims abstract description 6
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 claims abstract description 6
- 230000006798 recombination Effects 0.000 claims abstract description 6
- 238000005215 recombination Methods 0.000 claims abstract description 6
- 230000014509 gene expression Effects 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 28
- 238000000034 method Methods 0.000 claims description 14
- 238000005516 engineering process Methods 0.000 claims description 13
- 238000012546 transfer Methods 0.000 claims description 12
- 230000009261 transgenic effect Effects 0.000 claims description 11
- 239000000969 carrier Substances 0.000 claims description 8
- 230000029087 digestion Effects 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 238000000338 in vitro Methods 0.000 claims description 7
- 210000000130 stem cell Anatomy 0.000 claims description 6
- 241000894006 Bacteria Species 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 230000009465 prokaryotic expression Effects 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000012216 screening Methods 0.000 claims description 4
- 238000011156 evaluation Methods 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 108091000041 Phosphoenolpyruvate Carboxylase Proteins 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract description 3
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000011990 functional testing Methods 0.000 abstract 3
- 238000000746 purification Methods 0.000 abstract 1
- 210000001161 mammalian embryo Anatomy 0.000 description 16
- 210000001519 tissue Anatomy 0.000 description 12
- 241001465754 Metazoa Species 0.000 description 10
- 235000013601 eggs Nutrition 0.000 description 10
- 230000004927 fusion Effects 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 108020004414 DNA Proteins 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 210000000287 oocyte Anatomy 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 210000001672 ovary Anatomy 0.000 description 5
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 4
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 4
- 239000011630 iodine Substances 0.000 description 4
- 229910052740 iodine Inorganic materials 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 3
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 210000001015 abdomen Anatomy 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 210000003754 fetus Anatomy 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000035800 maturation Effects 0.000 description 3
- 239000002480 mineral oil Substances 0.000 description 3
- 235000010446 mineral oil Nutrition 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 210000004508 polar body Anatomy 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000012797 qualification Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 2
- 229920000089 Cyclic olefin copolymer Polymers 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000005662 Paraffin oil Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013339 cereals Nutrition 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000002158 endotoxin Substances 0.000 description 2
- 230000001605 fetal effect Effects 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 235000003642 hunger Nutrition 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 230000002611 ovarian Effects 0.000 description 2
- 230000035479 physiological effects, processes and functions Effects 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000001742 protein purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000037351 starvation Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000011830 transgenic mouse model Methods 0.000 description 2
- 230000010474 transient expression Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 238000009736 wetting Methods 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- 101000942941 Arabidopsis thaliana DNA ligase 6 Proteins 0.000 description 1
- 108091033409 CRISPR Proteins 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 239000012901 Milli-Q water Substances 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 238000010459 TALEN Methods 0.000 description 1
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000001467 acupuncture Methods 0.000 description 1
- 210000001557 animal structure Anatomy 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002459 blastocyst Anatomy 0.000 description 1
- 210000001109 blastomere Anatomy 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000012930 cell culture fluid Substances 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000006059 cover glass Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 101150024923 da gene Proteins 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000280 densification Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- AAOVKJBEBIDNHE-UHFFFAOYSA-N diazepam Chemical compound N=1CC(=O)N(C)C2=CC=C(Cl)C=C2C=1C1=CC=CC=C1 AAOVKJBEBIDNHE-UHFFFAOYSA-N 0.000 description 1
- 229960003529 diazepam Drugs 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 235000013861 fat-free Nutrition 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 230000003328 fibroblastic effect Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000002394 ovarian follicle Anatomy 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 235000015277 pork Nutrition 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- QTENRWWVYAAPBI-YCRXJPFRSA-N streptomycin sulfate Chemical compound OS(O)(=O)=O.OS(O)(=O)=O.OS(O)(=O)=O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O.CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](N=C(N)N)[C@H](O)[C@@H](N=C(N)N)[C@H](O)[C@H]1O QTENRWWVYAAPBI-YCRXJPFRSA-N 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 229940099259 vaseline Drugs 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to genetic engineering, a kind of CPP5 Cre fusion protein functional tests and its application for being used to reject marker gene is specifically disclosed, the CPP5 Cre fusion protein functional tests include 5 short amino acid cell-penetrating peptide CPP5（KLPVM）With site differential recombination enzyme Cre albumen.After being incubated using the CPP5 Cre fusion protein functional tests after expression and purification with the pig fibroblast with riddled basins, somatic cell clone is carried out after obtaining the positive colony that riddled basins are rejected, the transgene pig that the riddled basins in F0 generations are rejected is obtained.
Description
Technical field
The present invention relates to genetic engineering, specifically, it is related to a kind of fusion protein for being used to reject marker gene and its answers
With.
Background technology
Animal Transgenic Technology is on the basis of classical genetics, molecular genetics and DNA recombinant techniques, with gene
The technological means such as engineering, external source target gene interested or recombination are stably integrated in the chromogene of host animal
Group and a kind of emerging biometric technology for stably entailing offspring.Most earlier than 1976 by Jaenisch first with retrovirus
Infecting mouse body early embryo obtains Mohs leukemia virus transgenic mice first.With the appearance of new technologies, animal
Transgenic technology is also among constantly improving.Utilize traditional transgenic method, such as microinjection, sperm-mediated gene transfer, electricity
Perforation method, somatic cell clone method etc., have been prepared for a variety of transgenic animals such as transgenic mice, rat, pig, ox, sheep, chicken.
And because pig in the mankind has a large amount of similar dissections, physiology, metabolism, pathology.For example, they have a closely similar stomach
Enteron aisle is dissected and function, pancreas form and Metabolism regulation.Therefore pig is particularly suited for human disease model's research than mouse, simultaneously
Pork is the main source of nutrition of the mankind again.Along with the appearance of the state-of-the-art technologies such as ZNF/TALEN/Cas9 so that transgenosis is big
Animal has more wide application prospect, such as prepares animal bioreactor, improves Production of Livestock and Poultry ability and meat milk quality, training
Educate disease-resistant animal, production the mankind with animal organ, set up human disease model etc., huge economic benefit will be brought to the mankind.
In China, main herding veriety then depends critically upon import, and in main herding veriety, dairy bread degree of dependence reaches
100%, pig, chicken breed degree of dependence are close to 90%, it means that as the herding veriety in the whole industrial chain source of animal agricultural,
Depend critically upon international market.Therefore transgenic technology improve in the urgent need to.The barrier that maximum limitation transgenic technology develops at present
Hinder be riddled basins presence.Because in transgenic protocol, in addition to expressing target gene, in addition it is also necessary to selection markers base
Cause.Marker gene is that selected marker refers to that transformed cells can be made in genetic transformation（Or individual）From numerous non-turn
Change the marker gene screened in cell.Because transgene efficiency is low, it is therefore necessary to positive colony is entered using marker gene
Row screening and enrichment.But the presence of marker gene not only produces influence to contiguous gene and its destination gene expression, while will also
Bring serious safety issue.Although having some technologies to remove riddled basins at present, these methods are main
For mouse, and there is very big defect.Zymophore is for example recombinated by transient expression Cre, it is necessary to extra cell transfecting process,
And there is integration hidden danger and cytotoxicity.Seldom in the research of big animal, 2009, this seminar utilized in-vitro transcription mRNA
Transient expression Cre enzymes, obtain the transgenic cow that marker gene is knocked out.So far, riddled basins also are not carried out to pig
I.e.（marker free）Correlative study.Therefore easy, efficient, the transgene pig technology of Fast Labeling gene knockout is set up to grind
Study carefully and be particularly important.
The content of the invention
In order to solve problems of the prior art, it is used to reject marker gene it is an object of the invention to provide a kind of
Fusion protein and its application.
In order to realize the object of the invention, egg is merged present invention firstly provides a kind of CPP5-Cre for being used to reject marker gene
In vain, the fusion protein includes cell-penetrating peptide CPP5 and site differential recombination enzyme Cre albumen, the cell-penetrating peptide CPP5's
Amino acid sequence is as shown in sequence table SEQ ID No.1.
Further, the amino acid sequence of the fusion protein is as shown in sequence table SEQ ID No.2.
Present invention also offers a kind of carrier for expressing foregoing CPP5-Cre fusion proteins.
Present invention also offers a kind of method for rejecting transgene pig riddled basins, methods described includes following step
Suddenly：
1）Build CPP5-Cre prokaryotic expression carriers；
2）Utilize step 1）Expression vector carries out the expression and purifying of CPP5-Cre fusion proteins；
3）By step 2）Obtained CPP5-Cre fusion proteins are co-cultured with the porcine somatic cell with riddled basins；
4）The positive colony that evaluation and screening marker gene is rejected；
5）In F0 generations, are obtained by somatic cell clone, the transgene pig individual that riddled basins are rejected.
Further, the step 1）By the primer pair containing two restriction enzyme sites of CPP5 sequences and its NcoI and NdeI,
By making linker in vitro；PET28.2-Cre is cut with NcoI+NdeI enzymes are double, recovery obtains purpose fragment and is connected with linker,
Convert, shake bacterium, small upgrading grain, the pCPP5-CRE carriers of the digestion identification positive；The nucleotide sequence of the pCPP5-CRE carriers
As shown in sequence table SEQ ID No.3.
Further, the step 3）By step 2）Obtained CPP5-Cre fusion proteins are with carrying riddled basins
Pig fibroblast, be incubated co-culturing and is cloned.
Further, the step 5）By step 4）Obtained positive colony cell is screened as nuclear transfer donor cell,
In vitro egg mother cell is nuclear transfer recipient cell, and carrying out somatic cell clone by nuclear transfer technology obtains F0 for selection markers base
Because of the transgenic positive pig of rejecting.
Sieved invention further provides foregoing CPP5-Cre fusion proteins and the carrier for expressing the fusion protein in production
Select the application in the transgene pig of marker gene rejecting.
The beneficial effects of the present invention are：Transgene pig selection markers are removed the invention provides easy, efficient, quick
Genetic method is marker free.Its principle is the cell-penetrating peptide and site differential recombination enzyme Cre/ using 5 short amino acid
The albumen of loxP system constructings fusion, which makes it have, to be worn film, enters core, shearing marker gene function.As long as will purifying
The albumen gone out is incubated after 3h with the pig fibroblast with marker gene, and co-cultivation is cloned, and then enters performing PCR mirror
It is fixed, positive colony is used for later stage nuclear transfer, you can marker free transgene pig is obtained in F0 generations.
The present invention solves the technical problem of pig marker gene knockout, for following application of big Animal Transgenic and its base
Important basis is laid in plinth research.
Brief description of the drawings
Fig. 1 contains the fusion protein expression vector figure of short cell shuttle peptide and Cre enzymes for the present invention；
Fig. 2 is CPP5-Cre protein purifications figure of the present invention；
Fig. 3 is acquisition positive colony PCR qualification results after albumen of the present invention processing；
Fig. 4 is marker free positive F0 of the present invention for pig PCR testing results；
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Carrier design is completed by this seminar in embodiment, and sequencing is completed by Beijing Hua Da gene.Taq enzyme, T4DNA
Ligase, restriction endonuclease are purchased from Beijing NEB companies, and somatic cell clone agents useful for same is purchased from Sigma companies.Digestion, connection, return
The normal experiment operating procedures such as receipts, conversion, PCR amplifications, prokaryotic expression and its purifying are referred to《Molecular cloning（The third edition）》.Implement
Fusion protein expression vector figure of the example 1 containing short cell shuttle peptide and Cre enzymes
5 amino acid short peptide CPP5 are building up to pET28.2-Cre prokaryotic expression carriers（Addegene companies provide）In.
Synthesized first by Sheng Gong companies and contain two digestions of CPP5 sequences and its NcoI and NdeI in 1 pair of primer, primer
Site：
Up:catgggcAAACTGCCGGTTATGggcca；
Down:tatggccCATAACCGGCAGTTTgcc。
Then by making linker, 50 systems in vitro：Each 22.5 μ l of primer, 5 μ l 10xbufferPCR.99 degree of denaturation
5 minutes room temperature renaturation 10 minutes, 4 degree preserve with testing later.
PET28.2-Cre is cut with NcoI+NdeI enzymes are double, system is 50 μ l：The μ l of carrier 2, each 2 μ l, 10xbuffer4 of enzyme add 5
μ l, sterilized water 31 μ l, 37 degree of digestion 2h, run the purpose fragment that glue reclaim obtains 6319bp；By the fragment of above-mentioned recovery and
Linker connections, the μ l of system 10：Reclaim fragment 7 μ l, big linker 1 μ l, 10xbuffer and add 1 μ l, T4 ligase 1 μ l, 16 degree
Connect overnight.
Convert, shake bacterium, small upgrading grain, the pCPP5-CRE carriers of the digestion identification positive（Nucleotide sequence such as SEQ ID
Shown in No.3）, it is standby.
The CPP5-Cre protein purification figures of embodiment 2
1.CPP5-Cre prokaryotic expression
1）BL21 bacterium containing expression vector are inoculated into 5ml culture medium, 37 DEG C of incubated overnights；
2）1:100 are forwarded in 500ml culture medium, 37 DEG C of culture 2.5h；
3）Add IPTG to final concentration 0.2mM, 16 DEG C of induction 16h.
2.Ni-Nta posts are purified
1）Collect bacterium, add the solutionA of culture volume 1/10, be resuspended and sonicated cells；
2）13000g centrifuges 10min, and supernatant is with 0.45 μm of membrane filtration；
3）The milli Q water washings of 5 times of volumes of Ni posts, then balanced with the solutionA of 5 times of volumes；
4）Loading；
5）After sample has all flowed, pillar is rinsed with the solutionA of 8 times of volumes；
6）8 times of volume solutionA+ wash pillar；
7）Albumen is washed from pillar with the solution B of 3 times of volumes；
8）Albumen is replaced into the DMEM of 10% glycerine with ultrafiltration post.
3. remove endotoxin
1）Endotoxin gel is gone to add in post bed 1ml, natural subsidence；
2）0.2N NaOH detergent gels and immersion overnight with 5 times of volumes；
3）With the NaCl column scrubbers bed of 5 times of volumes；
4）5 times of volume MilliQ water washings post beds；
5）With 5 times of glycerine DMEM column scrubbers beds of volume 10%；
6）Loading, collects efflux；
7）With 2 times of glycerine DMEM column scrubbers beds of volume 10%, efflux is collected；
8）Packing and jelly are in -80 DEG C of preservations after step 3.2 to 3.4 step actifier column bed, protein determination concentration.
Positive colony PCR qualification results are obtained after the processing of the albumen of embodiment 3
1st, long white fetal fibroblast is set up
1）Take and fallopian tubal uterus, wrapping outlet are taken after pregnant 30d Landrace, execution, transport laboratory back in 2h.
Fetus is taken out from intrauterine, fetus is cleaned with the DPBS containing antibiotic, is transferred in superclean bench, use eye scissors
Remove fetus head, four limbs, internal organ, rinsed with DPBS.
2）Remainder is tried one's best with eye scissors in diameter 100mm Tissue Culture Dish and shredded.
3）A little serum is added, 1ml pipette tips tip is cut with scissors, it is about 40mm above sections to leave diameter, is connected
1ml rifles, tissue block are transferred to the bottom wall of 3 T25 Tissue Culture Flasks, are uniformly spread out tissue block with elbow straw.
4）The one of tissue block will be covered with upwardly, it is each to add 5ml cell culture fluids, it is put into CO2Incubator, 39 DEG C, 5%CO2、
100% humidity culture.
5）After after culture 6-8h, the one side for being covered with tissue block is overturn, cell culture fluid is submerged tissue block.
6）Whether culture has cell to climb out of in 5 days or so around tissues observed block.
7）When cell growth to 80% is converged, carry out Secondary Culture or carry out freezen protective.
2nd, recombinant protein and fibroblast are incubated and then turned out clone
The CPP5-Cre recombinant proteins expressed in embodiment 2 are incubated with long white fetal fibroblast and cultivated.By 5um-
The CPP5-Cre albumen of 10um concentration is added, and in the fibroblastic culture medium of cellar culture pig, then carries out cell culture, is made
After 3-24 hours, then PBS changes the cell culture medium without CPP5-Cre recombinant proteins, then carries out unicellular culture
Cloned, identified with PCR later after 2 weeks.
3rd, albumen removes positive colony PCR after marker gene and identified
Previously obtained cloned segment cell cryopreservation, part cell are extracted into genome and enter performing PCR identification（PCR points
Sub- qualification figure is as shown in Fig. 3-A）, identification original singly strike the amplification of two primers of pig primary cell M1 and M2 can expand 2374bp and
Two fragments of 1102bp, 664bp and 1102bp two can be expanded after marker gene is deleted, then with two primer amplifications of M1 and M2
Fragment, when in PCR amplification procedures, when extension of time is set to 45s, expands not 2374bp bands.Go out Fig. 3-B and show PCR amplifications
As a result, wherein 2,4,5,7,9,11,12,14,15,17 and 18 amplifications remove 664bp and 1102bp bands, it was demonstrated that be that marker gene is deleted
The positive colony removed,）, for follow-up cloning experimentation.
Embodiment 4marker free positive F0 are for pig PCR testing results
1st, in-vitro maturity of porcine oocytes
Ovary is taken from slaughterhouse, 28 DEG C -35 DEG C are put into, in the physiological saline containing penicillin and streptomycin sulphate, transported in 2h
Return the ovarian follicle of 3-6mm on 20mL syringes suction ovary of the use for laboratory equipped with No. 18 syringe needles.Extract is put in 50mL centrifugations
Guan Zhong, 37 DEG C of water-baths stand 15min and remove supernatant, add PVA-TL-HEPES and precipitation is resuspended, then stand 15min, are repeated once, will
Re-suspension liquid is put into diameter 60mm plastic culture dish, under Stereo microscope, and ovarian cumulus is selected with mouth suction pipe and wraps up more than 2 layers,
Densification, and the uniform cumulus cell-oocyte complex of kytoplasm（Cum μ Lus-Oocyte-complexes, COCs）, use
Maturation culture solution is washed 3 times and is transferred in the culture drop that 4h has at least been balanced in incubator（Every 100 μ L drops put 25 pieces）.
4 drops are done in each diameter 35mm plastic culture dish, are covered with embryo's level mineral oil.COCs is first in maturation culture solution
20 ± 2h is cultivated, is then transferred into no hCG and eCG ripe liquid and continues to cultivate 20 ± 2h.
2nd, body cell prepares
Using serum starvation method, by the target practice positive cell obtained in embodiment 3 when it grows to 80% and converged, carry out
Serum starvation processing, i.e., the FBS in nutrient solution（Gibco,Life Technologies）Concentration is down to 0.5% continuation from 20%
2-5d is cultivated, is digested according to a conventional method, centrifuge washing, finally adds 1mL micromanipulations liquid that cell precipitation is resuspended standby.
3rd, receptor oocytes stoning and donorcells nuclear transfer
Born of the same parents are selected after carrying out mature oocyte stoning, i.e. maturation 40-44h porcine oocytes, de- ovarian cumulus using blind suction method
Matter is uniform, and perivitelline is clear, and the complete egg mother cell of after birth is put into no Ca2+、Mg2+, NCSU-23 is standby is transferred to for HEPES bufferings
In micromanipulation drop：1h is carried out before nuclear transfer, diameter 60mm Tissue Culture Dish lid center（Drop 50-80 μ l, 2-3mm is wide, 8-
10mm length, paraffin oil covering）, act on 15-30min, donorcells and mature oocyte and meanwhile be transferred to wherein in 39%,
5%CO2, 100% wetting balance 15min, micro- fixing pipe and stoning/injection needle are installed, operating system position is regulated, in dress
Under inverted microscope equipped with micromanipulation instrument and thermostatic platform, 40 × use fixing pipe（25-35 μm of internal diameter, 100-120 μm of external diameter）
Sticking egg mother cell stirs egg mother cell with stoning/injection needle of 15-25 μm of internal diameter, first polar body is in 1 o'clock of clock and watch position
Put 200 × under, from 3 points of clock and watch at oolemma is crossed into stoning/injection acupuncture, suction out first polar body and its nearby a little cytoplasm moved back
Go out stoning/injection needle, first polar body and a little kytoplasm are spued and select 15-20 μm of diameter, refractivity is strong, it is circular, it is smooth
Body cell, 200 × lower with stoning/injection needle one donorcells of absorption, body cell is expelled to oolemma at stoning inserting needle
Under perivitelline in press oolemma with injection needle, make the cell membrane of donorcells and acceptor ooecium matter intimate contact with one another every batch
After the completion of 25-30 egg mother cell, structure, after terminating by donorcells-ooecium texture into cell pair（Reconstructed eggs）It is transferred to
In NCSU-23+4mg/ml BSA, 39 DEG C, 5%CO2, repair 1-2h in 100% humidified incubator.
4th, fusion and activation
The reconstructed eggs recovered are transferred in fusion liquid in batches and balance 3min, are washed with fusion/activation liquid after 3 times, often
Batches 5 are put into and have been paved with the integration slot of fusion liquid, with drawing and tip very thin solid glass pin stirs recombinant eggs, make
Donorcells-recipient oocyte film contact surface and electrode runs parallel ECM2001 fusion instruments apply the straight of one 30 μ s, 2.0kv/cm
The electric pulse induced fusion of stream is activated simultaneously, is washed with NCSU-23%+4mg/ml BSA 5 times, the embryo of mineral oil covering is transferred to immediately
In tire nutrient solution, 39 DEG C, 5%CO2, take out after 100% humidity culture 0.5h~1h, the decision fusion under stereomicroscope.
5th, Embryo Culture
At least 4h well in advances culture drop before micromanipulation is started, 35mm Micro-Organism Culture Dish is taken in super-clean bench, is done
6-8 drops big 30 μ L, are carefully added into the covering of 2.5-3ml mineral oil, CO are put into after carrying out mark2Incubator inner equilibrium will be upper
State fusion reconstructed embryo wash 5 times with embryo medium after be transferred to, each 30 μ L 8-10 reconstructed embryo of drop culture, culture
The spilting of an egg and Blastocyst formation result are recorded during 48h and 168h.
6th, embryo transfer
With boar examination feelings or observation pressure back of the body reaction, spontaneous estrus sow is selected, typically will on the day of structure clone's embryo
In CO2The 1-2 cell clones embryo that 12-30h is cultivated in incubator takes out, and loads in 2.5ml tubules.With the tinfoil paper paper wrapper of sterilizing
It is good, mark is carried out, is put into constant temperature transport case and transports to pig transplanting place, use 40-60ml（1mg/100kg body weight）Physiology salt is water-soluble
Solve 1-1.5mg pentothals and diazepam, ear vein injection 20ml, after the preliminary fiber crops of acceptor pig after, lifted on operation bracket
Lie on the back（Young replacement gilt）Or lie on one's side（Suprapubic arch sling）Baoding is to replacement gilt：The 1st pair and the 2nd pair of nipple below belly
Between, the local 15 × 10cm of ventrimeson2First iodine disinfection after cleaning shaving in area, rear alcohol swab takes off iodine（To Suprapubic arch sling：
Belly the part between the ribs and the hips nest 15 × 10cm of portion2In the range of clean, alcohol takes off iodine after shaving iodine disinfection）Prepare moving knife cut ventrimeson it
Before, above remaining arcotic is taken the circumstances into consideration again injection 20-30ml, cut a mouth for being about 8-10cm along ventrimeson, open abdomen
Chamber, is covered after visiting a hand of entering, taking-up ovary with the sterilized non-fat gauze of wetting（And to Suprapubic arch sling:One is done along flank portion
The individual otch that is about 10cm vertical with ventrimeson face）Ovary will be taken out in surgical staff, will when adjusting fimbriae tubae
Embryo in suction pipe, which takes out, to be placed in thermal station, is loaded embryo in grafts under stereomicroscope, surgical staff and embryo
Transplanting personnel are mutually coordinated, and grafts are probeed into fallopian tubal at least 5cm from fimbriae tubae portion substantially combines to ampulla-isthmus
Place, one side pushing syringe removes whether stereoscopic Microscopic observation embryo after grafts transplanting is still trapped in grafts, really so as to outer
Recognize without after, start ovary return, 10d intramuscular injection 800IU hCG, 13d injection 500IU after art mouthful suture embryo transfer
PMSG。
7th, blastomere is counted
Reconstructed embryo blastaea is taken out, after fixing 10min after being washed 3 times in the DPBS containing 3.7% paraformaldehyde again by fixation
Blastaea is transferred to containing 10 μ g/ml Hoechst33342（bisbenzimide）DPBS in lucifuge magazine in be incubated at room temperature
After 10min dyeing terminates, blastaea is transferred on slide, as far as possible carrying liqs less, as early as possible with 9:1 vaseline/paraffin oil exists
Four posts of its four angle point, covered, the careful pressing cover glass under stereomicroscope makes ovum somewhat be expanded after being pressurized but not
It is advisable as broken, uses and observe, take a picture under nail sheet for oil seal, NikonE800 microscopes, ultraviolet excitation, counting.
Embodiment 5F0 is for boar positive identification
Extractions of the F0 for porcine tissue genomic DNA：
The boar that pig farm is born（Positive cell clone in embodiment 3）A small amount of tail tissue block is taken, is shredded
Or cell precipitation is put into 1.5ml centrifuge tubes, plus 700 μ l DNA extraction buffers, 20 μ l Proteinase Ks（20mg/ml）, mix 56
DEG C water-bath digestion more than 18h, until tissue block or cell precipitation are wholly absent, interval shakes to obtain more preferable effect, adds
700 μ lTris- saturated phenols, gently shake 12min, and 12000 rpm centrifuge 12 min.By upper phase be transferred to it is another it is new from
In heart pipe, previous step is repeated, upper phase is transferred in another new centrifuge tube, add 700 μ l phenol:It is imitative（1:1）, gently
Shake 12min, 12000rpm centrifugations 12min.Upper phase is transferred in another new centrifuge tube, 700 μ l chloroforms, temperature are added
With shake 12 min, 12000rpm centrifuges 12 min.Upper phase is transferred in another new centrifuge tube, plus 2 times of volumes without
Water-ethanol, is overturned after mixing, and 12000rpm centrifuges 10 min.Supernatant is abandoned, is washed and precipitated and tube wall with 70% ethanol, tipping ethanol,
Drip raffinate to the greatest extent, room temperature lower open mouth places 20~30min, ethanol is volatilized completely, plus in right amount（About 100 μ l）TE buffer solutions, in 56
Detected under dissolving DNA in DEG C water-bath, 0.7% agarose gel electrophoresis detection genomic DNA concentration, measurement 260/280nm wavelength
The concentration and purity of genomic DNA, are adjusted to debita spissitudo, -20 DEG C of preservations, while it is positive public to enter performing PCR identification target practice
Pig.Enter row agarose gel electrophoresis detection genomic DNA, if that expand 800bp and 388bp bands is both marker free
The positive, if only expanding 800bp for wild type or singly striking the individual without place to go marker gene, testing result such as Fig. 4 institutes
Show, wherein wherein No. 1-12 12 F0 are marker free success individuals for pig, wherein WT is wild type and +/- singly struck
For control.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (4)
1. a kind of method for rejecting transgene pig riddled basins, it is characterised in that the described method comprises the following steps：
1) CPP5-Cre prokaryotic expression carriers are built；
2) utilize step 1) expression vector carry out CPP5-Cre fusion proteins expression and purifying；
3) by step 2) obtained CPP5-Cre fusion proteins and the pig fibroblast with riddled basins be incubated
Co-cultivation is cloned；
4) positive colony that evaluation and screening marker gene is rejected；
5) in F0 generations, are obtained by somatic cell clone, the transgene pig individual that riddled basins are rejected；
The CPP5-Cre fusion proteins include cell-penetrating peptide CPP5 and site differential recombination enzyme Cre albumen, and the cell is worn
Film PEPC PP5 amino acid sequence is as shown in sequence table SEQ ID No.1.
2. according to the method described in claim 1, it is characterised in that the step 1) will containing CPP5 sequences and its NcoI and
The primer pair of two restriction enzyme sites of NdeI, by making linker in vitro；PET-28.2CRE is cut with NcoI+NdeI enzymes are double, is reclaimed
Obtain purpose fragment to be connected with linker, convert, shake bacterium, small upgrading grain, the pCPP5-CRE carriers of the digestion identification positive；It is described
The nucleotide sequence of pCPP5-CRE carriers is as shown in sequence table SEQ ID No.3.
3. according to the method described in claim 1, it is characterised in that the step 5) by step 4) screen obtained positive colony
Cell is nuclear transfer recipient cell as nuclear transfer donor cell, in vitro egg mother cell, and it is thin to carry out body by nuclear transfer technology
Born of the same parents clone obtains the transgenic positive pig that F0 is rejected for riddled basins.
Application of the 4.CPP5-Cre fusion proteins in the transgene pig that production riddled basins are rejected, it is characterised in that will
CPP5-Cre fusion proteins are co-cultured with the pig fibroblast with riddled basins, what evaluation and screening marker gene was rejected
Positive colony, F0 generations are obtained by somatic cell clone, the transgene pig individual that riddled basins are rejected；
The CPP5-Cre fusion proteins include cell-penetrating peptide CPP5 and site differential recombination enzyme Cre albumen, and the cell is worn
Film PEPC PP5 amino acid sequence is as shown in sequence table SEQ ID No.1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410158270.5A CN103992409B (en) | 2014-04-18 | 2014-04-18 | Fusion protein and its application for rejecting marker gene |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410158270.5A CN103992409B (en) | 2014-04-18 | 2014-04-18 | Fusion protein and its application for rejecting marker gene |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103992409A CN103992409A (en) | 2014-08-20 |
| CN103992409B true CN103992409B (en) | 2017-08-25 |
Family
ID=51306770
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410158270.5A Expired - Fee Related CN103992409B (en) | 2014-04-18 | 2014-04-18 | Fusion protein and its application for rejecting marker gene |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103992409B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107177630A (en) * | 2017-05-09 | 2017-09-19 | 华南农业大学 | A kind of anti-PCV2 transgene pigs preparation method without exogenous marker gene |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8183342B2 (en) * | 2007-11-06 | 2012-05-22 | Case Western Reserve University | Method of treating chemotherapy-induced thrombocytopenia |
| CN102827873A (en) * | 2012-07-31 | 2012-12-19 | 西北农林科技大学 | Cre fusion protein functional test cell and modified Cre fusion protein |
-
2014
- 2014-04-18 CN CN201410158270.5A patent/CN103992409B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8183342B2 (en) * | 2007-11-06 | 2012-05-22 | Case Western Reserve University | Method of treating chemotherapy-induced thrombocytopenia |
| CN102827873A (en) * | 2012-07-31 | 2012-12-19 | 西北农林科技大学 | Cre fusion protein functional test cell and modified Cre fusion protein |
Non-Patent Citations (1)
| Title |
|---|
| Cell-Penetrating Penta-Peptides (CPP5s): Measurement of Cell Entry and Protein-Transduction Activity;Jose A.Gomez et al.;《Pharmaceuticals (Basel)》;20101212;第3卷(第12期);摘要，第9页第2段、最后一段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103992409A (en) | 2014-08-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104419719B (en) | A kind of method that transgene pig riddled basins are knocked out | |
| CN105821049B (en) | A kind of preparation method of Fbxo40 gene knock-out pig | |
| CN106191064B (en) | A method of preparing MC4R gene knock-out pig | |
| CN105087620B (en) | One kind is overexpressed the 1BB carriers of pig costimulation acceptor 4 and its application | |
| CN103725710B (en) | One oneself can delete free carrier and application thereof | |
| CN107937345B (en) | A kind of fibroblastic method of pig for preparing while knocking out CD163 gene and CD13 gene | |
| CN113957069B (en) | Composition for simultaneous modification of amino acids at 736 th site and 738 th site of pAPN gene and application thereof | |
| CN104263754A (en) | Reconstructed ovum of albinism model pig and construction method thereof, and construction method of model pig | |
| CN101195833B (en) | Low-temperature incubation transgene method for cultivated silkworm diapause breed variety | |
| CN103993027B (en) | A kind of method that transgene pig riddled basins are knocked out | |
| CN113957093B (en) | System for site-directed modification of pAPN gene and application thereof | |
| CN105524940A (en) | Vector, cell and method for improving bovine cloning efficiency on the basis of histone methylation modifying level | |
| CN113604504A (en) | Composition for site-directed modification of pAPN gene 16 exon and application thereof | |
| CN108410894A (en) | A kind of carrier and method improving ox cloning efficiency based on histone methylated horizontal modification | |
| CN101260398A (en) | Nerve growth factor gene positioning reconstruction animal and its preparation method and application | |
| CN103468732A (en) | Expression vector for piggyBac transposon, and transgenic pig and construction method thereof | |
| CN103992409B (en) | Fusion protein and its application for rejecting marker gene | |
| CN102517329A (en) | Method for breeding transgenic pig for over expression of pig PBD-2 gene | |
| CN104830752B (en) | A kind of unicellular cultural method of porcine fetus fibroblasts | |
| CN104894163B (en) | A kind of method and application of the non-human mammal preparing the dual-gene knockout of GGTA1 and iGb3S | |
| CN108611371A (en) | The preparation method of the Goats Milk gland bioreactor of GP5-M albumen is expressed in milk | |
| CN106086080B (en) | A method of ox cloning efficiency is improved using miRNA | |
| CN101956003A (en) | Method for detecting exogenous gene integration sites in pig genome | |
| CN101570763A (en) | Transgene pig for preparing animal model for human diseases and culturing method thereof | |
| CN107541484A (en) | Diploid red crucian caudal fin cell system 2nFC and its construction method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170825 |