CN106047926B - One kind turning 15 fatty acid desaturase gene pig of Δ 12 and Δ and its preparation method and application - Google Patents
One kind turning 15 fatty acid desaturase gene pig of Δ 12 and Δ and its preparation method and application Download PDFInfo
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Abstract
The invention discloses one kind to turn 15 fatty acid desaturase gene pig of Δ 12 and Δ and its preparation method and application, Δ 12 and 15 fatty acid desaturase gene fat2 and fat1 gene of Δ are inserted into No. 10 chromosome DCTN5 gene intron 2 of pig in the form of amalgamation and expression by transgenic method, acquisition turns fat1-fat2 fusion pig, the transgene pig respectively organize in great expression n-3 and n-6PUFAs, in conjunction with the health-care effect of PUFAs, the tissue such as pork with health role is produced using the transgene pig, applied in the treatment and prevention and treatment of human diseases.Since pig immune system and metabolism are quite similar with the mankind, the PUFAs of secretion will not generate immunological rejection in human disease treatment, compared to other similar transgenic animals more safety and efficiently, take in convenient for human foods.And drug is added relative to PUFAs external source, the intake toxic side effect of this transgenosis pork is less, can become external source PUFAs substitute of new generation.
Description
Technical field
The present invention relates to the animals and its preparation method and application obtained by transgenic method, in particular to one kind turns Δ
12 and 15 fatty acid desaturase gene pig of Δ and its preparation method and application.
Background technique
15 fatty acid desaturase gene of Δ 12 and Δ (fat2 and fat1) is polyunsaturated fatty acid in the mammalian body
Two kinds of key enzymes of (Polyunsaturated fatty acids, PUFAs) route of synthesis control n-6 and n-3PUFAs respectively
Synthesis rate.15 fatty acid desaturase gene of Δ (fat1) was initially found by Spychalla in 1997 for the first time, was then ground
Study carefully and shows that the gene can significantly improve n-3PUFAs content.And 12 fatty acid desaturase gene of Δ (fat2) is also demonstrate,proved under study for action
It is real that n-6PUFAs is obviously improved.PUFAs plays important physiological function to the growth and development of people.It is controlled respectively by fat1 and fat2
The n-3 and n-6PUFAs of synthesis nerve cell, stem cell differentiation with developmentally play an important role, DHA therein be considered and
The development of human nervous system is closely bound up.In addition to this, both PUFAs for cardiovascular disease, skeletal diseases, cancer and
Inflammation has improvement and therapeutic effect, is accepted extensively and be applied in a variety of therapeutic schemes by people.
For Δ 12 and 15 fatty acid desaturase gene of Δ (fat2 and fat1) is lacked in the mammalian body, lead to not
Itself normal synthesis n-3 and n-6PUFAs.Livestock is both needed to external source addition C18:3n-3 and C18 during the feeding process at present:
2n-6 is to meet the original substrate accumulation of two kinds of PUFAs route of synthesis, convenient for DHA direction composition.Due in the mammalian body
Lack enough DHA and restrict its growth and development, a variety of products rich in DHA emerge in succession and obtain health medicine lot number.People
Affirmative and demand for DHA health benefit are considerably beyond its yield.
It attempts to turn 15 fatty acid of Δ 12 or Δ by the production of the transgenic animals such as pig, ox, sheep there are many research at present
Delta 8 desaturase genes (fat2 and fat1), but these transgenic animals can only solve 15 fatty acid desaturase gene of Δ 12 or Δ
One of (fat2 and fat1) in other words can only promote one of n-3 or n-6PUFAs route of synthesis, can not mention simultaneously
High n-3 and n-6PUFAs content, it is palliative.For this status, existing team prepares while turning 15 fat of Δ 12 and Δ
The zebra fish of sour delta 8 desaturase genes (fat1 and fat2 gene), can improve n-3 and n-6PUFAs content simultaneously really.But its at
Fruit disadvantage is: 1) transgenic zebrafish can not be applied at once.Zebra fish is model organism for studying from using there are one
Set a distance；2) to husbandry sector without directive function.Fish and mammal on PUFAs is synthesized there are larger difference, fish at
Fruit can not apply in husbandry sector, need to also make a search again in mammal level；3) its dual-gene takes is total to 2A sequence
Expression-form is attached expression so that the expression quantity of two genes is there are larger difference, especially 3 ' end gene expression amounts compared with
It is low.
In consideration of it, being highly desirable in husbandry sector and human health research to 15 fatty acid desaturation of Δ 12 and Δ
Enzyme gene (fat2 and fat1) clone and amalgamation and expression, and preparation turns fat1-fat2 fusion pig, so that the transgene pig
Pork in express 15 fatty acid desaturase of Δ 12 and Δ simultaneously.
Summary of the invention
The object of the present invention is to provide the pig that one kind turns 15 fatty acid desaturase gene of Δ 12 and Δ (fat2 and fat1),
Δ 12 and 15 fatty acid desaturase gene fat2 and fat1 gene of Δ are inserted into the form of amalgamation and expression by transgenic method
In No. 10 chromosome DCTN5 gene intron 2 of pig, acquisition turns fat1-fat2 fusion pig, the transgene pig each group
Equal great expression n-3 and n-6PUFAs in knitting can have using transgene pig production and protect in conjunction with the health-care effect of PUFAs
The pork etc. of strong effect, applied in the treatment and prevention and treatment of human diseases.Due to pig immune system and metabolism and the mankind ten
Seemingly, the PUFAs of secretion will not generate immunological rejection in human disease treatment to split-phase, compared to other similar transgenosis
Animal is more safe and efficient, takes in convenient for human foods.And drug, this transgene pig are added relative to PUFAs external source
The intake toxic side effect of meat is less, and becomes external source PUFAs substitute of new generation.
According to an aspect of the invention, there is provided one kind turns 15 fatty acid desaturase gene pig of Δ 12 and Δ, the Δ
12 and 15 fatty acid desaturase gene pig of Δ genome in include 15 fatty acid desaturase of Δ 12 and Δ fusion
Sequence, the fusion gene sequence of 15 fatty acid desaturase of the Δ 12 and Δ is as shown in SEQ ID NO:1, and the transgene pig
Pork be rich in n-3 and n-6PUFAs.The fat1-fat2 fusion gene sequence as shown in SEQ ID NO:1 is transferred to as a result,
In gene pig, so that transgene pig production is rich in the pork of n-3 and n-6PUFAs, people can be by eating outside pork mode
Source obtains n-3 and n-6PUFAs, and the status of PUFAs is lacked so as to improve mankind itself.In conjunction with the health-care effect of PUFAs, prevention and treatment
Human diseases.In addition, people absorb external source PUFAs by way of food pork can reduce moment supplement PUFAs health care product
Status, reduce health care product uptake, reduce human body toxic side effect, meet to greatest extent people for growth and development institute it is required
The dosage of PUFAs.
In some embodiments, the fusion gene sequence of 15 fatty acid desaturase of the Δ 12 and Δ is by 15 fatty acid of Δ
Delta 8 desaturase genes (fat1 gene) and 12 fatty acid desaturase gene of Δ (fat2 gene) are directly connected to composition fat1-fat2
Fusion.Fat2 gene and fat1 gene share a promoter as a result, and the two is neighbouring to connect into fat1-fat2 fusion base
Cause after the fusion is integrated into the chromosome of transgene pig, is started by promoter CAG and is transcribed, then translate into a fusion
Albumen is expressed, but this albumen has the function of 15 fatty acid desaturase of 12 fatty acid desaturase of Δ and Δ, and two kinds simultaneously
The intimate equivalent of enzyme respectively plays effect and its function.Thus, it is possible to which integrating a fat1-fat2 by transgenic technology merges table
Up on gene to the chromosome of transgene pig, and the amalgamation and expression gene plays 15 rouge of 12 fatty acid desaturase of Δ and Δ simultaneously
The effect of fat acid desaturase improves the health care and edible value of the transgene pig and its meat.
In some embodiments, this turns No. 10 dyeing that fat1-fat2 fusion gene sequence is incorporated into transgene pig
On body.
In some embodiments, this turns No. 10 dyeing that fat1-fat2 fusion gene sequence is incorporated into transgene pig
On body in DCTN5 gene intron 2.DCTN5 gene is the known open gene (GenBank on No. 10 chromosome of pig
Accession number:NC_010452.3), and DCTN5 gene region is active for gene expression on No. 10 chromosome
Area.As a result, on No. 10 chromosome that fat1-fat2 fusion is incorporated into transgene pig in DCTN5 gene intron 2
When, which can correctly express fat1-fat2 fusion, and PUFAs in the musculature (pork) of the transgene pig
Content highest produces the pork rich in n-3 and n-6PUFAs, improves the status for lacking PUFAs in pork, and people eat the pig
The effect of preventing and treating a variety of diseases can be played after meat.
According to another aspect of the present invention, a kind of system for turning 15 fatty acid desaturase gene pig of Δ 12 and Δ is provided
Preparation Method, the transgene pig are by fat1-fat2 amalgamation and expression gene integration into pig genome, in pig by transgenic technology
Great expression in musculature (pork) generates n-3 and n-6PUFAs abundant.In conjunction with the health-care effect of PUFAs, people are being eaten
The status that mankind itself lacks PUFAs can be improved when with the transgenosis pork, prevent and treat human diseases.PUFAs can be reduced simultaneously
The demands status of health care product reduces health care product to the secondary possibility of human body poison, and meeting people to greatest extent must for growth and development
Need the dosage of PUFAs.The transgene pig obtained by this method, pork are rich in n-3 and n-6PUFAs, and yield is high, will not be right
The mankind generate immunological rejection.Seem more safe and efficient compared to other similar transgenic animals, this method includes as follows
Step:
(1) transgenic fusion expression vector pCAG-fat1-fat2-EGFP is constructed, which includes fat1-
Fat2 amalgamation and expression gene order, CAG promoter sequence, EGFP sequence；
(2) the transgenic fusion expression vector built is transfected into porcine fetus fibroblasts, after drug screening, obtained
Positive pig cell；
(3) the positive pig cell that screening obtains is obtained into clone embryos after somatic cell nuclear transfer technique, by clone embryos
The intrauterine for migrating to receptor sow cultivates primary transgene pig；
(4) the primary transgene pig of birth is detected, hereditary Δ 12 and 15 rouge of Δ can be stablized in offspring by filtering out
Fat acid delta 8 desaturase genes, and the transgene pig of PUFAs is rich in pork.
In some embodiments, the gene order of transgenic fusion expression vector pCAG-fat1-fat2-EGFP such as SEQ
Shown in ID NO:2.
In some embodiments, transgenic fusion expression vector pCAG-fat1-fat2-EGFP is incorporated into described
On No. 10 chromosome of transgene pig in DCTN5 gene intron 2.
In some embodiments, porcine fetus fibroblasts are boar fetal fibroblast, primary turn produced
Gene pig is transgenosis boar.Target gene is transfected using boar fetal fibroblast as target cell, and is filtered out positive thin
Born of the same parents carry out body-cell neucleus transplanting as the donor cell of nuclear transfer using this positive cell and obtain clone embryos, clone embryos are moved
It plants to the intrauterine of receptor sow, cultivates primary transgenosis boar.It is carried out as a result, using this primary transgenosis boar as male parent
Breeding can largely obtain the offspring pig that can stablize hereditary Δ 12 and 15 fatty acid desaturase gene of Δ.When primary transgenosis
When pig is boar, due to not influenced by period of pregnancy etc., there is stronger reproductive capacity than sow.
According to the third aspect of the present invention, it provides and a kind of turns answering for Δ 12 and 15 fatty acid desaturase gene pig of Δ
With the preparation method of the application comprising the pork rich in 15 fatty acid desaturase of Δ 12 and Δ turns from there through establishing to utilize
Gene pig production can be widely applied to the mankind in conjunction with the health-care effect of PUFAs rich in the method for n-3 and n-6PUFAs pork
It is safer, more efficient in the treatment and prevention and treatment of disease, it comprises the following steps:
1) 15 fatty acid desaturase gene pig of Δ 12 and Δ is turned according to above method preparation；
2) turn Δ 12 and 15 fatty acid desaturase gene pig of Δ using what is obtained in step 1) as male parent and breed, obtain
Heredity must be stablized and turn 15 fatty acid desaturase gene of Δ 12 and Δ, and be rich in the transgene pig of PUFAs in pork；
3) by step 2) turn Δ 12 and 15 fatty acid desaturase gene pig of Δ is butchered, obtain be rich in PUFAs
Pork.
In some embodiments, the pork rich in PUFAs is applied in human health care as pork product, is mankind's disease
Disease plays preventive and therapeutic effect.
Detailed description of the invention
Fig. 1 is to turn fat1-fat2 fusion gene carrier structural schematic diagram.
Fig. 2 is to turn fat1-fat2 fusion expression vector PCR testing result: 1,2 be EGFP gene PCR amplification in figure；3,4
For fat1-fat2 fusion PCR amplification；" M " is Marker label.
Fig. 3 is the positive transgenic cell fluorescence testing result after drug screening: " Bright field " is indicated in white light
Lower control cell；" Blue Light field " indicates cell under blue light illumination and expresses green fluorescence.
Fig. 4 is the fluorescence results figure of primary transgene pig: " Normal light " be denoted as under white light transgene pig and its
Tissue control；" Blue light " mark transgene pig expresses green fluorescence under blue light illumination.
Fig. 5 is that primary transgene pig PCR detects electrophoresis result figure: " H in figure2O " indicates blank control；" Neg. " indicates non-
Transgene pig；" Pos. " indicates positive vector control；" M " indicates Marker label；"Transgenic pigs(935800-
935802) each primary transgene pig individual number " is indicated；" Fat1fat2gene " is the fat1-fat2 fusion being transferred to；
" EGFP gene " is green fluorescence protein gene；" GADPH gene " is pig reference gene.
Fig. 6 is the Southern blot result figure of primary transgene pig: " M " indicates Marker label in figure；"0.5c-
2c " indicates 3 positive copy gradients；" Transgenic pigs (935800-935802) " indicates each primary transgene pig
Body number.
Fig. 7 is the mRNA relative expression quantity testing result of fat1-fat2 gene in primary transgene pig musculature: " WT "
Indicate non-transgenic pig；" 935800-935802 " indicates each primary transgene pig individual number；" β-actin " is indicated in pig
Join gene.
Fig. 8 fat1-fat2 gene mRNA relative expression quantity difference between each transgene pig individual different tissues:
" 935800-935802 " indicates each primary transgene pig individual number；Figure A is musculature；Figure B is skin histology；Scheming C is
Adipose tissue.
Fig. 9 is that fat1-fat2 fusion gene sequence is incorporated into the comparison on No. 10 chromosome of No. 935801 transgene pigs
Result analysis chart.
Specific embodiment
The invention will now be described in further detail with reference to the accompanying drawings.
Pst-fat1 plasmid origin is in Chinese Academy of Sciences Guangzhou Institute of Biomedicine and Health；PcDNA3.1 carrier is purchased from
Excellent precious biology；PEGFP-N1 empty carrier is purchased from excellent precious biology；Fat2 gene order derives from GenBank accession
Number:NM070159；Fat1 gene order derives from GenBank accession number:178291.
1, fat1-fat2 fusion gene carrier and its PCR amplification detection are constructed.
Fat1 gene is amplified from pst-fat1 plasmid using round pcr, fat2 gene is announced according to GenBank
Sequence direct labor synthesis.Using overlap extension pcr, will be connected as between fat1 the and fat2 gene after amplification
Fat1-fat2 fusion (as shown in SEQ ID NO:1) shares a promoter after the neighbouring connection of fat1 and fat2 gene
CAG, transcription and translation is fusion protein, and this fusion protein can play the function and fat2 gene of fat1 gene simultaneously
Function.CMV, NEO, bGH-polyA sequence are expanded from design primer on pcDNA3.1 carrier simultaneously.Using digestion interconnection technique,
Fat1-fat2 fusion and a series of synthetic genes are connected to pEGFP-N1 carrier, which contains EGFP gene,
Transgenic animals and cell screening can be done.Fat1-fat2 integrative gene expression vector: pCAG-fat1- is completed in final building
Fat2-EGFP (as shown in SEQ ID NO:2).Fusion expression vector overall length 15300bp.CMV promoter starts EGFP gene
Express green fluorescence；CAG promoter starts target gene fat1-fat2 amalgamation and expression.Its structure map is shown in attached drawing 1.
Respectively on plasmid EGFP gene and fat1-fat2 fusion carry out PCR amplification inspection, band is clearly single
One, as a result correctly, amplification is shown in attached drawing 2.
2, positive transgenic cell fluorescence detects.
Fat1-fat2 integrative gene expression vector is imported into boar fetal fibroblast using liposomal transfection teclmiques,
Cell transient expression green fluorescence.After G418 is screened 20 days, remaining cell is the Green fluorescent colonies group of stable transfection, positive
Cell fluorescence testing result is shown in attached drawing 3.
3, turn the preparation and its identification of fat1-fat2 fusion pig.
Cell clone to express green fluorescence after screening is rolled into a ball as donorcells, by body-cell neucleus transplanting method, 3
Head sow goes up 868 pieces of clone embryos of co-transplantation.2 sows return feelings, and 1 farrowing sow gives birth to 3 young offspring boars living, this 3
The ID number of head transgene pig is respectively 935800,935801,935802.120 day age of progeny transgenic pig carries out butchering survey respectively
Determine content of polyunsaturated fatty acid.
The genomic DNA of 3 transgene pig ear tissues (is purchased from Omega by Tissue DNA extraction kit
Company) kit is stripped.Three pairs of primer pairs are respectively adopted in Fat1-fat2 gene, EGFP gene and pig reference gene GADPH
It carries out corresponding PCR amplification detection.Fusion fat1-fat2, EGFP gene, internal reference all can be detected in 3 transgene pigs
The presence of gene GADPH, band is single, and length is respectively 1942bp, 2118bp, 182bpPCR, and PCR testing result is shown in attached drawing 5.
Using portable fluorescent lamp, the transgene pig green fluorescence expression in 3 day age is detected.It visually observes visible 3 and turns base
It is transgenic positive pig because pig has green fluorescence label.Green fluorescence detection is carried out again at 120 day age of transgene pig,
The expression of transgene pig green fluorescence is stablized.The green fluorescence of transgene pig body surface is concentrated mainly on transgene pig snout, hoof
Expression.It being found after dissection sampling, the shallower group of the colors such as heart, tongue of primary transgene pig is woven with apparent green fluorescence,
Attached drawing 4 is shown in transgene pig fluorescence detection.
3, integration mode and expression of the foreign gene in different transgene pigs compare.
Appropriate transgene pig genomic DNA is extracted, Southern blot detects the integration feelings of fat1-fat2 fusion
Condition.There are two kinds of Integration Modes for the approving and forwarding gene pig as the result is shown, wherein 935800 and 935802 have 3 hybridization positive bands；
935801 hybridize positive band for single, thus it is speculated that 935800 and 935802 come from the same clone cell group, generate 3 and integrate position
Point is greater than 2 copies in single integration site；And 935801 are originated from another cloning cluster, only one integration site, purpose base
Because that may be integrated into single allele, copy number is about 2 copies.The results are shown in attached figure 6 by the Southern blot of transgene pig.
Detect the expression quantity discovery of transgene pig different times different tissues mRNA, 120 day age transgene pig (935800-
935802) there is fat1-fat2 gene mRNA expression in, the results are shown in attached figure 7.No. 935802 transgene pigs are in flesh in 120 day age
Expression quantity is minimum in meat, fat and skin histology.935800 and No. 935801 transgene pigs are No. 935802 turns in musculature
4.36 times of gene pig mrna expression amount and 3.94 times, the results are shown in attached figure 8 A；In skin histology, 935800 and 935801 mRNA
Expression quantity is 2.65 times and 3.58 times of 935802, as a result sees Fig. 8 B；Adipose tissue is similar to skin histology expression quantity, and 935800
Mrna expression amount with 935801 is 1.91 times and 2.30 times of 935802, as a result sees Fig. 8 C.
Due to 935800 suitable with the expression quantity of 935801 transgene pigs fat1-fat2 gene mRNA in each tissue,
And only one copy number of fat1-fat2 gene in 935801 transgene pigs, it is more stable in the heredity of offspring, therefore
Select the transgene pig with for further study.
4, foreign gene integrates the analysis of position in No. 935801 primary transgene pig genomes.
In order to determine integration position of the foreign gene in transgene pig genome, test is analyzed using inverse PCR technique
No. 935801 primary transgene pigs integrate foreign gene position.Design primer, by Inverse PCR products carry out sequencing and it is right
Sequencing result compares discovery with pCAG-fat1-fat2-EGFP carrier and pig genome sequence respectively, this turns fat1-fat2 fusion
Gene order is incorporated on No. 10 chromosome of the transgene pig in DCTN5 gene intron 2.DCTN5 gene is pig
Known open gene (GenBank accession number:NC_010452.3) on No. 10 chromosomes, and where the gene
Region is gene expression active region, and comparison result is shown in attached drawing 9.
5, No. 935801 transgene pig family transgenic Swine muscle PUFAs content analysis.
It is bred using No. 935801 primary transgene pigs as male parent, and constructs No. 935801 transgene pig familys, and
Be further analyzed, detection family transgenic pig respectively organize in content of polyunsaturated fatty acid.And dissect 120 day age 935801
Transgene pig in number transgene pig family samples muscle, fat, skin samples (about 10g) respectively, is put into -80 DEG C of ice rapidly
It is saved in case.It takes petroleum ether method to extract polyunsaturated fatty acid in tissue sample, entrusts China Guangzhou Analysis &. Test Center, according to
It is detected according to organic mass spectrometry method general rule (JY/T 003-1996), is as a result calculated using GC/MS area normalization method.
At 120 day age, select 3 transgene pigs from No. 935801 transgene pig familys, and select on the same day age 3 non-turn base
Because the musculature of pig is detected, n-3PUFAs total amount improves 41.84% compared with non-transgenic pig.Wherein C18:3n-3, C20:
5n-3, C22:5n-3 and C22:6n-3 content are respectively increased 40.38%, 133.33%, 33.33% and 130.00%.Simultaneously
C22:4n-6 and C20:4n-6 content increases by 9.52% and 84.09%.N-6PUFAs total amount is improved compared with non-transgenic pig
33.36%.N-6/n-3PUFAs ratio is adjusted downward to 12.28 from accounting 13.06 in transgene pig, declines up to 5.97%, as a result sees
Following table:
Turn fat1-fat2 fusion pig as a result, and fat1 and fat2 fusion is integrated by pig by transgenic technology
In genome, heredity that can be stable in the offspring of transgene pig, and the great expression in Swine muscle (pork), it generates rich
Rich n-3 and n-6PUFAs.In conjunction with the health-care effect of PUFAs, people can improve mankind itself when eating the transgenosis pork
Lack the status of PUFAs, prevents and treats human diseases.
Above-described is only some embodiments of the present invention.For those of ordinary skill in the art, not
Under the premise of being detached from the invention design, various modifications and improvements can be made, these belong to the protection scope of invention.
Claims (4)
1. a kind of preparation method for turning 15 fatty acid desaturase gene pig of Δ 12 and Δ, which is characterized in that the preparation method
It comprises the following steps:
(1) transgenic fusion expression vector pCAG-fat1-fat2-EGFP is constructed, which includes fat1-fat2
Amalgamation and expression gene order, CAG promoter sequence, EGFP sequence, the fat1-fat2 amalgamation and expression gene order such as SEQ ID
Shown in NO:1；
(2) the transgenic fusion expression vector built is transfected into porcine fetus fibroblasts, after drug screening, obtained positive
Pig cell；
(3) the positive pig cell that screening obtains is obtained into clone embryos as donor cell after somatic cell nuclear transfer technique, it will
Clone embryo transplantation cultivates primary transgene pig to the intrauterine of receptor sow；
(4) the primary transgene pig of birth is detected, hereditary Δ 12 and 15 fatty acid of Δ can be stablized in offspring by filtering out
Delta 8 desaturase genes, and the transgene pig of PUFAs is rich in pork.
2. the preparation method according to claim 1 for turning 15 fatty acid desaturase gene pig of Δ 12 and Δ, feature exist
In the gene order of the transgenic fusion expression vector pCAG-fat1-fat2-EGFP is as shown in SEQ ID NO:2.
3. the preparation method according to claim 2 for turning 15 fatty acid desaturase gene pig of Δ 12 and Δ, feature exist
In the transgenic fusion expression vector pCAG-fat1-fat2-EGFP is incorporated into No. 10 dye of the transgene pig
In the intron 2 of DCTN5 gene on colour solid.
4. the preparation method according to claim 1 for turning 15 fatty acid desaturase gene pig of Δ 12 and Δ, feature exist
In the porcine fetus fibroblasts are boar fetal fibroblast, and the primary transgene pig is transgenosis boar.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206675A (en) * | 2011-03-23 | 2011-10-05 | 中国农业科学院北京畜牧兽医研究所 | Novel vector for raising content of polyunsaturated fatty acids in animal body |
| CN104059881A (en) * | 2013-03-21 | 2014-09-24 | 中国农业大学 | Method for producing polyunsaturated fatty acid-containing transgenic animal |
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2016
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102206675A (en) * | 2011-03-23 | 2011-10-05 | 中国农业科学院北京畜牧兽医研究所 | Novel vector for raising content of polyunsaturated fatty acids in animal body |
| CN104059881A (en) * | 2013-03-21 | 2014-09-24 | 中国农业大学 | Method for producing polyunsaturated fatty acid-containing transgenic animal |
Non-Patent Citations (1)
| Title |
|---|
| Double Transgenesis of Humanized fat1 and fat2 Genes Promotes Omega-3 Polyunsaturated Fatty Acids Synthesis in a Zebrafish Model;Shao-Chen Pang，et al;《Mar Biotechnol》;20141231;580-593页 |
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