CN105255889A - RNAi fragment, RNAi vector, preparation method and application of RNAi vector - Google Patents
RNAi fragment, RNAi vector, preparation method and application of RNAi vector Download PDFInfo
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- CN105255889A CN105255889A CN201510639871.2A CN201510639871A CN105255889A CN 105255889 A CN105255889 A CN 105255889A CN 201510639871 A CN201510639871 A CN 201510639871A CN 105255889 A CN105255889 A CN 105255889A
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Abstract
The invention provides an RNAi fragment, an RNAi vector, a preparation method of the RNAi vector and application of the RNAi vector and relates to the technical field of biology. The RNAi fragment is used for interfering Zfy genes on sheep chromosomes Y and characterized in that the sequence of the RNAi fragment is shown in SEQ ID NO.1 or SEQ ID NO.2; protein, in charge of encoding, of the Zfy genes is used as a transcription factor and is related to occurrence of sperms. The RNAi fragment can block or hurt or damage normal development and functions of sperms Y by silencing the Zfy genes in the germ cell sperms Y of male sheep.
Description
Technical field
The present invention relates to biological technical field, be specifically related to preparation method and the application thereof of a kind of RNAi interference fragment, RNAi interference carrier and RNAi interference carrier.
Background technology
Under field conditions (factors), nearly all mammiferous sex ratio all close to 1:1, thus achieves and stably produces offspring without interruption, is issued to the eubiosis in natural condition.For husbandry sector is actual, desirable sex ratio tool is of great significance.Utilize lactation, manufacturing enterprise that egg-laying deseription carries out producing wish to obtain more female individuals, and produce meat, fibre trait at male
upper tablenow obtain dominance more obvious.All the time, the mankind just have great interest to the sex controlling animal offspring.Current sex control is mainly carried out from prefecundation and latter two aspect of fertilization, and the former is fertilized after applying spermatozoa isolation again, makes the sex just determining offspring at the time of fertilization; The latter adopts Early-stage judgment, carries out the method for the sex screening of embryo to control the sex of offspring.
Mammal sex determination theory is thought, sex chromosome is that the zygote of " XX " just can develop into female individuals, and will developing into that sex chromosome is " XY " is male.So fundamentally, controlling the optimal method of Livestock Sex is first be separated X, y sperm, and then is fertilized, and just can control the sex of offspring artificially, this is approach simple, the most most economical in sex control in theory.After the 1950's, the research report about spermatozoa isolation is a lot, and these researchs all try
figurethe physical property (volume, density, electric charge, mobility) utilizing X sperm different with y sperm), adopt the diverse ways such as centrifuging, electrophoretic method, ion exchange method, cell-surface antigens method to realize the separation of sperm, but have a common problem---repeatable poor.And these differences change along with the difference of envrionment conditions, therefore, many results of study are usually inconsistent, even occur contrary conclusion, cause two some Difference of class sperm to there is dispute.Up to the present, except X, y sperm DNA content have except difference can affirm, otherwise difference performance is very little, is even difficult to determine.But along with the development of Protocols in Molecular Biology, and the continuation of research work deeply, and new achievement in research will continue to bring out.
The principle that there are differences according to Mammals X, y sperm DNA content at present, utilize wandering cells to retrieve instrument and carry out separation X, y sperm, separating effect is best, purity can reach more than 90%, but due to apparatus expensive, the reasons such as velocity of separation is slow, few for the sperm quantity of being fertilized, vigor is poor, can not meet the heavy demand to sexing semen on producing far away.By contrast, if can find in two kinds of sperm developments or fertilization process, even critical X, Y chromosome special mRNA during fetal development, RNA is utilized to disturb (RNAInterference in conjunction with emerging Protocols in Molecular Biology in the present age, RNAi) method is then expected to lower cost, easy, realize to animal sex control rapidly.
RNAi is the gene silencing (Post-transcriptionalGeneSilencing after a kind of transcribing, PTGS) phenomenon, by the double-stranded RNA (Dubble-strandedRNA.dsRNA) that produces in external synthetic or body at the cell internal specific ground mRNA of its homology of degrading, make corresponding Gene interfere, realize the expression stoping target gene.RNAi method can lower the genetic expression in cell quick, easy, effectively, specifically, and it does not still study a kind of powerful of gene function, and treats for specific gene and provide new technique means, and its application prospect is very wide.
All prove in the research of genome, mRNA and protein level, X, y sperm genetic expression there are differences really.Genetic expression detect delay shows, at the meiotic pachytene of spermiogenesis tail, for preventing some enzymes and sex chromosome from having an effect, two sex chromosome high enrichment form gonosome.The formation of gonosome protects sex chromosome, and on sex chromosome, the expression of special gene is closed.Therefore, there is no transcribing of mRNA in mammalian mature sperm, but but containing mRNA in mammalian mature sperm, this species diversity is from transcribing in spermatogenesis, and its translation far lags behind transcribes.These mRNA instruct some necessary albumen of synthesis after spermioteleosis, or after fertilization supplementing ovocyte mRNA storehouse, or play a part key as RNAi to fertility and embryo growth and development.
Hendriksen etc. find the research of mouse, and in reduction division process, except Xist genetic expression, nearly all sex chromosome gene is not all expressed, but meiosis anaphase, some sex chromosome specific genes start to express.Postmeiotic, detects that on Y chromosome, Ubely and Sry gene has higher mRNA level in-site; In X sperm, Ubelx gene mRNA high level expression also detected, find the expression product of X chromosome specific gene Mhr6A simultaneously.Somebody finds the expression of X, other specific genes of Y chromosome in addition, as X chromosome specific gene Akap82 (skelemin of sperm tail) and Nap-X (a kind of core associated protein of encoding), Y chromosome specific gene Zfy-1, Zfy-2 and Y353/B.
Wherein Zfx/Zfy gene is the pair of alleles of encoding zinc finger protein on karyomit(e), and express from meiophase, the content in round spermatid is the highest.Zfx gene is a member in ZFY (zincfinger-Y) gene family, and ZFY family comprises Zfx, Zfy and Zfa gene, and they are closely similar on molecular structure.General Mammals has Zfx and Zfy gene, and Zfx gene is positioned on X sex chromosome, and be the gene that female, boar X chromosome must contain, Zfy gene is positioned on Y sex chromosome, and two genes are positioned at heterosomal end, but are not positioned at homology region.Zfx gene comprises one and acid transcribes exciting region, a nuclear localization sequence and a DNA joining region, has 13 C2H2 [Cys (2) His (2)] type zinc fingers.In mammalian cell, C2H2 zinc fingers is one of modal protein structure.Find that kind more than 800 has the protein of this zinc fingers at present, there is important physiological function.The albumen of Zfx/Zfy coding, as transcription factor, has some functions and relevant with the generation of sperm in spermiogenesis tail process.
Summary of the invention
In view of this, the embodiment of the present invention provides a kind of RNAi interference fragment, interference carrier, preparation method and application.Main purpose is disturbed the Zfy gene on animal Y chromosome by RNAi interference carrier, Zfy gene expression amount declined, reaches the object of manual control Animal Sex with this.
For achieving the above object, the present invention mainly provides following technical scheme:
A kind of RNAi interference fragment, for disturbing the Zfy gene on animal Y chromosome, described RNAi interference fragment sequence is for shown in SEQIDNO.1 or SEQIDNO.2;
The albumen of described Zfy negative gene responsible editor code is as transcription factor, relevant with the generation of sperm.
On the other hand, above-mentioned RNAi interference fragment is as the application of the medicine of preparation interference animal y sperm gene.
On the other hand, a kind of RNAi interference carrier, for disturbing animal y sperm gene, is characterized in that, described RNAi interference carrier clone has RNAi interference fragment, and the sequence of described RNAi interference fragment is for shown in SEQIDNO.1 or SEQIDNO.2.
Further, described RNAi interference carrier comprises pLentiLox3.7 carrier, and described pLentiLox3.7 carrier is connected with described RNAi interference fragment.
On the other hand, described RNAi interference carrier is carrying out applying in sex control animal prefecundation.
Further, described RNAi interference carrier is injected in animal body in the mode of testis direct injection, for disturbing the Zfy gene on animal Y chromosome.
A preparation method for RNAi interference carrier, comprises the steps:
(1) enzyme cuts pLentiLox3.7 carrier;
(2) the described pLentiLox3.7 carrier after being cut with enzyme by described RNAi interference fragment is connected, and obtains connecting product;
Wherein, described RNAi interference fragment sequence is for shown in SEQIDNO.1 or SEQIDNO.2;
(3) by described connection product conversion in competent cell, the bacterium liquid obtained also is carried out order-checking and identifies by screening, and sequencing result and the on all four bacterium liquid of described RNAi interference fragment sequence is increased;
Described restriction enzyme site is HpaI and XholI; Described hairpin structure sequence is for shown in SEQIDNO.3 or SEQIDNO.4;
(4) from the bacterium liquid of described amplification, RNAi interference carrier is extracted.
Further, described competent cell is pLentiLox3.7 carrier cell.
Further, described screening is to screen containing pLentiLox3.7 carrier, restriction enzyme site and hairpin structure.Compared with prior art, the present invention at least has following advantages:
The invention provides
?rNAi interference fragment, by carrying out silence to the Zfy gene in the sexual cell y sperm of male sheep, hindering, damaging or destroying normal development and the function of y sperm.With give X sperm during female sheep mating and to be more fertilized chance, effectively control the sex of sheep thus the ewe ratio that first filial generation sheep is produced raises greatly in prefecundation.Present method can not cause damage to required spermatid or reduce its vigor, and with low cost.
Accompanying drawing explanation
fig. 1for the expression level of ZfymRNA in cell in RNAi interference fragment of the present invention, interference carrier, preparation method and application thereof.
fig. 2for RNA in RNAi interference fragment of the present invention, interference carrier, preparation method and application thereof disturbs the female lamb rate in sheep Zfy first filial generation.
in figure: each group compares with control group, and supreme target is that difference is not remarkable; " * " represents significant difference (P<0.05).
fig. 3for the vector construction signal in RNAi interference fragment of the present invention, interference carrier, preparation method and application thereof
figure.
Embodiment
For further setting forth the present invention for the technique means reaching predetermined goal of the invention and adopt and effect, be described in detail as follows in conjunction with preferred embodiment.
Embodiment 1
Design and synthesize the RNAi interference fragment for disturbing the Zfy gene in animal testis androgone.The present embodiment is for the RNAi interference fragment of the Zfy gene in sheep testicle androgone below, is described technical scheme provided by the invention.Specific as follows:
Filter out two siRNA fragment according to mRNA sequences Design 8 siRNA fragment templates of sheep Zfy gene, wherein
table 1for the ZfycDNA template for siRNA;
table 1for the ZfycDNA template of siRNA
Then according to the requirement of described pLentiLox3.7 carrier and institute's tape starting, the oligonucleotide sequences of synthesis two couples of shRNAi.These two pairs of shRNAi interference fragments are respectively shRNAA and shRNAG,
as table 2shown in.
The Zfy gene order used comes from NCBI gene pool, filters out 8 siRNA fragment according to RNAi principle of design and the compare of analysis result on NCBI,
as table 1shown in, be the ZfycDNA template for siRNA.Add Xhol I and Hpa I restriction enzyme site in 5 ' and 3 ' of each siRNA fragment respectively, then synthesize siRNA fragment and filter out two pairs of shRNAi interference fragments.
table 2two pairs of shRNAi interference fragments
Embodiment 2
The screening method of pair shRNAi interference fragment of two described in embodiment 1 is specific as follows:
1, the RNAi interference carrier of Zfy gene is built:
The Zfy gene order used comes from NCBI gene pool, filters out 8 siRNA fragment according to RNAi principle of design and the compare of analysis result on NCBI,
as table 1shown in, be the ZfycDNA template for siRNA.Add Xhol I and Hpa I restriction enzyme site in 5 ' and 3 ' of each siRNA fragment respectively, then send biotech firm to synthesize siRNA fragment.Positive and negative adopted chain is combined into double-strand by annealing and is connected to plentilox3.7 (Addgene, the U.S.) on slow virus packaging system carrier, then be transformed into (Tiangen, China) in E.coliDH5 α competent cell to increase, extract plasmid-20 DEG C preservation.
2, the external interference test of Zfy gene:
Choose the male sheep testicle of health 2 in 1 years old age, 75% alcohol drenches sterilization, dual anti-PBS soaking flushing, and cell cultures is indoor, adopts two step enzyme digestions to isolate sustentacular cell of testis and androgone.These isolated cells are put into the HamF12/DMEM nutrient solution containing 10% foetal calf serum, interior interpolation HEPES (15mol/L), 100kU/L penicillin and 0.1g/L Streptomycin sulphate, 1ug/ml Urogastron, 100ulITS (Regular Insulin, Transferrins,iron complexes, sodium selenate) { Regular Insulin (10 μ g/ml), Transferrins,iron complexes (10 μ g/ml) }, 3.3 × 10
-7mol/L vitamin A acid, vitamin A (3.3 × 10
-7mo/L), 10 μ g/ml vitamin-Es, 10
-4mol/L vitamins C, 10
-3mol/L pyruvic acid, 10
-7mol/L testosterone, 25U/LRFSH.About by 1 × 10
6cELL/cm
2density be inoculated in and be placed with in six orifice plates of cover glass, at 32 DEG C, 5%CO
2, humidity be under the condition of 95% cultivate 24 hours, as transfection reagent, the carrier built is transfected in androgone with calcium ion, often organizes in triplicate.The total serum IgE of androgone is extracted in transfection after 48 hours, detect the mrna expression level of Zfy gene with RT-PCR.
as table 3be depicted as goal gene primer sequence and PCR condition.
table 3goal gene primer sequence and PCR condition
Pick out good three experiments carrying out next step of interference effect,
as table 4shown in, hereinafter referred to as PllA (Zfy1759), PllF (zfy1773) and PllG (Zfy1888),
table 4for the ZfyRNAi sequence with HpaI and XholI restriction enzyme site filtered out.
table 4the ZfyRNAi sequence with HpaI and XholI restriction enzyme site filtered out
3, the animal body internal interference test of Zfy gene:
The plasmid of extraction is added the dual anti-PBS solution of band to dilute, be then diluted to 8ml by the amount of 1.5mg/ pipe, packing is carried out to plasmid.Six healthy sheep rams are divided into three groups, first group of injection pLLA, second group of injection pLLF, the 3rd group of injection pLLG, use No. 12 puncture needles injections along the testis longitudinal axis close to epididymis 1/3rd place by the dosage of every sheep every side testis 1.5mg.During injection, marginal not penetrates the slow pumpback puncture needle in limit, after injection, is slowly extracted by puncture needle, and to the measure of injection orifice strict sterilization.Interval 10d injects once, injects 3 times altogether.After third time injection, 4d starts to gather ram seminal fluid, carries out artificial insemination immediately, and joins ewe and often organizes 200, registers and joins ewe overbit, Breeding date, carrying out statistical study to the sex ratio quantity of offspring.
Result is as follows:
1, RT-PCR detects the expression level of Zfy gene mRNA in androgone:
as Fig. 1shown in, in result display experimental group PLLA, PLLF, PLLG, the expression level of Zfy gene mRNA is all lower than control group (physiological saline group).Wherein PLLF, PLLG and control group comparing difference are significantly (P < 0.05); PLLA difference extremely significantly (P < 0.01).So tentatively choose PLLA, PLLF, PLLG as to the good shRNA fragment of zfy Gene interfere effect,
as table 3shown in.
2, the sex ratio of first filial generation sheep:
as Fig. 2shown in, result display experimental group PLLF, PLLG, the female lamb rate of first filial generation sheep and control group do not have difference (P > 0.05), and the female lamb rate of the first filial generation sheep of experimental group PLLA comparatively control group is high, significant difference (P < 0.05), female lamb rate can reach 76.50%, is significantly higher than control group 48.00%.
table 5for each group of offspring (first filial generation) male and female sheep quantity and female lamb rate.
table 5first filial generation lamb male and female data statistic
Note: each group compares with control group, and supreme target is that difference is not remarkable; " * " represents significant difference (P<0.05).
Embodiment 3
A kind of RNAi interference carrier, comprise sequence for the RNAi interference fragment shown in SEQIDNO.1, its preparation method, comprises the steps:
(1) enzyme cuts pLentiLox3.7 carrier;
(2) the described pLentiLox3.7 carrier after being cut with enzyme by described RNAi interference fragment is connected, and obtains connecting product;
Wherein, described RNAi interference fragment sequence is SEQIDNO.1;
(3) by described connection product conversion in competent cell, to screen containing pLentiLox3.7 carrier, restriction enzyme site and hairpin structure and the bacterium liquid obtained to be carried out order-checking qualification, and sequencing result and the on all four bacterium liquid of described RNAi interference fragment sequence are increased;
Described restriction enzyme site is HpaI and XholI; Described hairpin structure sequence is SEQIDNO.3;
(4) from the bacterium liquid of described amplification, RNAi interference carrier is extracted.
Wherein vector construction signal
figure is as Fig. 3shown in.
Embodiment 4
Be with the difference of embodiment 3, RNAi interference carrier in the present embodiment, comprise sequence for the RNAi interference fragment shown in SEQIDNO.2, the sequence in corresponding preparation process for hairpin structure that the RNAi interference fragment shown in SEQIDNO.1 and sequence are SEQIDNO.3 replace with sequence for the RNAi interference fragment shown in SEQIDNO.2 and sequence be the hairpin structure of SEQIDNO.4.
RNAi interference carrier wherein in embodiment 3 and embodiment 4 on the impact of sheep first filial generation female lamb rate and the method contrasted with the female lamb rate in normal first filial generation thereof and result as the screening process in embodiment 2.
The above, it is only preferred embodiment of the present invention, not any pro forma restriction is done to the present invention, although the present invention discloses as above with preferred embodiment, but and be not used to limit the present invention, any those skilled in the art, not departing from the scope of technical solution of the present invention, make a little change when the technology contents of above-mentioned announcement can be utilized or be modified to the Equivalent embodiments of equivalent variations, in every case be do not depart from technical solution of the present invention content, according to any simple modification that technical spirit of the present invention is done above embodiment, equivalent variations and modification, all still belong in the scope of technical solution of the present invention.
Claims (9)
1. a RNAi interference fragment, for disturbing the Zfy gene on animal Y chromosome, is characterized in that, described RNAi interference fragment sequence is for shown in SEQIDNO.1 or SEQIDNO.2;
The albumen of described Zfy negative gene responsible editor code is as transcription factor, relevant with the generation of sperm.
2. RNAi interference fragment according to claim 1 is as the application of the medicine of preparation interference animal y sperm gene.
3. a RNAi interference carrier, for disturbing animal y sperm gene, is characterized in that, described RNAi interference carrier clone has RNAi interference fragment, and the sequence of described RNAi interference fragment is for shown in SEQIDNO.1 or SEQIDNO.2.
4. RNAi interference carrier according to claim 3, is characterized in that, described RNAi interference carrier comprises pLentiLox3.7 carrier, and described pLentiLox3.7 carrier is connected with described RNAi interference fragment.
5. RNAi interference carrier according to claim 4, is characterized in that, described RNAi interference carrier is carrying out applying in sex control animal prefecundation.
6. RNAi interference carrier according to claim 5, is characterized in that, described RNAi interference carrier is injected in animal body in the mode of testis direct injection, for disturbing the Zfy gene on animal Y chromosome.
7. a preparation method for RNAi interference carrier, is characterized in that, comprises the steps:
(1) enzyme cuts pLentiLox3.7 carrier;
(2) the described pLentiLox3.7 carrier after being cut with enzyme by described RNAi interference fragment is connected, and obtains connecting product;
Wherein, described RNAi interference fragment sequence is for shown in SEQIDNO.1 or SEQIDNO.2;
(3) by described connection product conversion in competent cell, the bacterium liquid obtained also is carried out order-checking and identifies by screening, and sequencing result and the on all four bacterium liquid of described RNAi interference fragment sequence is increased;
Described restriction enzyme site is HpaI and XholI; Described hairpin structure sequence is for shown in SEQIDNO.3 or SEQIDNO.4;
(4) from the bacterium liquid of described amplification, RNAi interference carrier is extracted.
8. the preparation method of RNAi interference carrier according to claim 7, is characterized in that, described competent cell is pLentiLox3.7 carrier cell.
9. the preparation method of RNAi interference carrier according to claim 7, is characterized in that, described screening is to screen containing pLentiLox3.7 carrier, restriction enzyme site and hairpin structure.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399308A (en) * | 2016-07-22 | 2017-02-15 | 石河子大学 | RNA interference fragment of zfy gene, expression vector and applications of RNA interference fragment and expression vector |
| CN107267511A (en) * | 2017-06-27 | 2017-10-20 | 石河子大学 | RNAi interference fragments, interference carrier and its preparation method and application |
| CN109735542A (en) * | 2019-01-24 | 2019-05-10 | 石河子大学 | RNAi interference fragment, interference carrier and its preparation method and application |
| CN109735630A (en) * | 2019-01-05 | 2019-05-10 | 兰州大学 | The detection method and label application of sheep ZFY gene mononucleotide polymorphism label |
| CN115806986A (en) * | 2022-10-28 | 2023-03-17 | 石河子大学 | Milk goat Zfy gene interference fragment, expression vector and application thereof |
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| CN102206676A (en) * | 2011-03-24 | 2011-10-05 | 石河子大学 | Vector for controlling gender of animals and application thereof |
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| 刘帅兵: "绵羊Zfy基因干扰载体筛选及性控效果的验证", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106399308A (en) * | 2016-07-22 | 2017-02-15 | 石河子大学 | RNA interference fragment of zfy gene, expression vector and applications of RNA interference fragment and expression vector |
| CN107267511A (en) * | 2017-06-27 | 2017-10-20 | 石河子大学 | RNAi interference fragments, interference carrier and its preparation method and application |
| CN107267511B (en) * | 2017-06-27 | 2020-06-16 | 石河子大学 | RNAi interference fragment, interference vector, preparation method and application thereof |
| CN109735630A (en) * | 2019-01-05 | 2019-05-10 | 兰州大学 | The detection method and label application of sheep ZFY gene mononucleotide polymorphism label |
| CN109735542A (en) * | 2019-01-24 | 2019-05-10 | 石河子大学 | RNAi interference fragment, interference carrier and its preparation method and application |
| CN115806986A (en) * | 2022-10-28 | 2023-03-17 | 石河子大学 | Milk goat Zfy gene interference fragment, expression vector and application thereof |
| CN115806986B (en) * | 2022-10-28 | 2024-04-16 | 石河子大学 | Milk goat Zfy gene interference fragment, expression vector and application thereof |
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