CN104694569A - Method for building animal fertilized eggs for milk micro-RNA function study - Google Patents
Method for building animal fertilized eggs for milk micro-RNA function study Download PDFInfo
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- CN104694569A CN104694569A CN201510079093.6A CN201510079093A CN104694569A CN 104694569 A CN104694569 A CN 104694569A CN 201510079093 A CN201510079093 A CN 201510079093A CN 104694569 A CN104694569 A CN 104694569A
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Abstract
The invention relates to a method for building animal fertilized eggs for milk micro-RNA function study and belongs to the technical field of biology. The method comprises the following steps: firstly, building MMTV-let-7g plasmids, and then building fertilized eggs of let-7g transgenic mice with milk gland specificity overexpression. The method for building the animal fertilized eggs for milk micro-RNA function study is conducive to completion of milk components, especially experimental materials for milk micro-RNA function study; and a reliable platform is provided for milk micro-RNA in vivo function study.
Description
Technical field
The present invention is specifically related to a kind of method setting up fertilised non-human eggs for the functional study of milk microRNA, belongs to biological technical field.
Background technology
Milk is newborn and baby stage important source of nutrition, wherein has abundant nutritive substance.In recent years, found have microRNA to exist in milk.MicroRNA be a class by the mode of degrade specific mRNA or the translation of arrestin matter on post-transcriptional level to the small molecules non-coding RNA (microRNA, miRNA) that genetic expression plays regulatory role.The non-coding microRNA of miRNA to be a class length be 19-25 Nucleotide, its effect extensively, can participate in regulating a series of important biological processes in vital movement.One of family the most deep is studied by Let-7 family in miRNA, it is regulated and controled by post-transcriptional level, the expression of interference target gene, thus participates in regulating cell growth, differentiation, apoptosis, the generation of tumour and multiple physiology and the pathologic process such as to cancerate.Let-7 is identified in nematode by Reinhart etc. first, finds that it participates in growing timing and regulates.The research such as Neilson also shows, in each cell of different developmental phases, the expression sum of miRNA is different, and namely expressing at cytocerastic different steps miRNA is dynamic regulation.So far, let-7g there is no report to mammary gland development and lactation function point analysis, significant to the research of microRNA function in milk.
At present, when studying a certain concrete microRNA, usually adopting the strategy of " process LAN ", namely by making specific microRNA content in cell or animal body increase someway, then by the phenotype that observation of cell, animal etc. occur, analyzing the function of this microRNA.As, adopt the miRNA of synthetic that cell is carried out to transfection, carries out intravenous injection or artificial feeding to animal.Its application has obvious deficiency: cost is higher, needs repeatedly to inject, and action time is short and unstable.In addition, the functional study for milk microRNA composition is difficult to be undertaken at experience card by cell levels experiment, and existing experimentation on animals cannot realize the experimental study for whole process lactation.Therefore current experiment material cannot meet some physiological and pathological mechanism, the needs of especially milk component functional study.
For the in depth function of microRNA in body research milk, we set up a kind of transgenic mice of mammary gland-specific process LAN microRNA, can this microRNA of process LAN in milk steadily in the long term.This mammary gland-specific process LAN microRNA mouse can be used for studying the effect that in milk, specific microRNA is grown filial generation mouse growth, can judge the physiological function of this microRNA in milk at body.Thus provide basic data for the Transformation Application of microRNA in follow-up milk, and as: add as quality of dairy products monitoring mark and milk-product nucleic acid.
Summary of the invention
The object of the present invention is to provide one to have Absorbable organic halogens to express, for the animal platform in body research milk microRNA physiological function.
In order to achieve the above object, the technique means that the present invention adopts is:
Set up a kind of method of the fertilised non-human eggs for the functional study of milk microRNA, it is characterized in that: comprise the following steps:
(1) MMTV-let-7g plasmid is built: the flanking sequence choosing people let-7g sequence and each 150bp of upstream and downstream thereof on NCBI, the cloning primer of design containing EcoR I restriction enzyme site, take genome as template, through the above-mentioned sequence of pcr amplification, EcoR I is the single endonuclease digestion PCR primer obtained and the pBluescript II carrier containing mammary gland specific promoter MMTV-LTR respectively, CIP is to carrier dephosphorylation, after purifying, through T4 ligase enzyme, the two is connected, be transformed into competent cell DH5 α after connecting product order-checking correctly, set up recombinant plasmid MMTV-let-7g;
(2) zygote of mammary gland special process LAN let-7g transgenic mice is set up: with the above-mentioned recombinant plasmid of Spe I single endonuclease digestion, running gel reclaims the microinjection transgenosis component obtaining purifying, this component comprises mammary gland specific promoter MMTV-LTR, Let-7g and transcription termination signal; The transgenosis component of acquisition purifying is imported through microinjection in the zygote survived.
In the present invention, preferably, the carrier 3h of EcoR the I PCR primer that obtains of single endonuclease digestion and the pBluescript II containing mammary gland specific promoter MMTV-LTR respectively during described structure MMTV-let-7g plasmid, CIP, to carrier dephosphorylation 1h, obtains 5 μ g products after purifying, through T4 ligase enzyme, the two is connected, competent cell DH5 α 50 μ L is transformed into, coated plate, next day after connecting product order-checking correctly, picking 5 bacterium colonies carry out order-checking qualification, determine recombinant plasmid MMTV-let-7g.
In the present invention, preferably, the described cloning primer containing EcoR I restriction enzyme site is: 7g-CL-F:aatgagaattctccaaatgtggtg, 7g-CL-R:ccttgaattcatcttcaaacttcttg.
In the present invention, preferably, the concentration of the transgenosis component described in adjustment is used for microinjection to 1ng ~ 2ng/uL.
Compared with prior art, beneficial effect of the present invention is embodied in:
The foundation that the present invention is used for the fertilised non-human eggs of milk microRNA functional study contributes to improving milk component, and the experiment material of especially milk microRNA functional study, for milk microRNA provides reliable platform in body functional study.
Accompanying drawing explanation
Fig. 1 is that microinjection transgenosis builds schematic diagram;
Fig. 2 be wild-type (WT) with let-7g content in transgenic (TG) mammary gland of mouse tissue compare schematic diagram; Wherein, the histogram on the left side represents let-7g content in wild-type (WT) mammary gland of mouse tissue, and the histogram on the right represents let-7g content in transgenic (TG) mammary gland of mouse tissue;
Fig. 3 is the generation mice body weight effect diagram of let-7g gene overexpression to lactation.
Embodiment
Below by experiment, also the present invention will be further described in conjunction with the embodiments, it should be understood that these embodiments only for the object of illustration, never limit the scope of the invention.
Concrete technical scheme of the present invention is:
1, the structure of MMTV-let-7g plasmid
Choose the flanking sequence of people let-7g sequence (reference sequences: NT_022517) and each 150bp of upstream and downstream thereof on NCBI, the cloning primer of design containing EcoR I restriction enzyme site, primer is: 7g-CL-F:aatgagaattctccaaatgtggtg, 7g-CL-R:ccttgaattcatcttcaaacttcttg.Take genome as template, through its sequence of pcr amplification, EcoR I is the single endonuclease digestion PCR primer obtained and the pBluescript II carrier 3h containing mammary gland specific promoter MMTV-LTR respectively, CIP, to carrier dephosphorylation 1h, obtains 5 μ g products, the two is connected through T4 ligase enzyme after purifying, competent cell DH5 α 50 μ L is transformed into after connecting product order-checking correctly, next day, picking 5 bacterium colonies, carry out order-checking qualification.Sequencing result shows, successfully establishes recombinant plasmid MMTV-let-7g (series connection of two copy precursor sequence).
2, the foundation of mammary gland-specific process LAN let-7g transgenic mice
With the above-mentioned recombinant plasmid of Spe I single endonuclease digestion, running gel reclaims the microinjection transgenosis component obtaining purifying.This component comprises mammary gland specific promoter MMTV-LTR, let-7g and transcription termination signal bGHpA (Fig. 1).Adjust the concentration of this component to 1ng ~ 2ng/uL.
B6 female mice is chosen, injection 5IU PMSG when the first day 13; The next day 12 time injection 5IU HCG, and to mate with male B6 mouse.The ICR male mice of oestrus Female ICR mice and vasectomy is mated simultaneously.Within 4th, will see that bolt B6 mouse cervical vertebra is from disconnected execution, dorsal position opens abdomen, and clip uterine tube nearly ovary end 0.5cm is placed in physiological saline.The embryo medium that whole uterine tube is placed in successively containing Unidasa is separated zygote, carries out vitro culture.Microinjection is carried out when 13.C57Bl/6 (B6) mouse, is commonly used to do for ovum mouse in transgeneic procedure.
By the zygote transplation survived after microinjection in the ampulla of uterine tube of ICR seeing bolt, in art, note the soft and insulation of operation.After 3 weeks, ICR mouse is given a birth.After suckling mouse is raw, 3w ear tag is its numbering, and clip about 0.5cm tail point carries out the qualification of foreign gene at genome conformity.ICR mouse, because its maternal instinct is comparatively strong, is used for the replace-conceive mouse that zygote feeds back.
3, the extraction of gDNA and screening
Mouse Tail-tip is placed in 0.5mL Digestive system, 55 DEG C of digestion 3h.Add equal-volume PCI, 1.5 × 10
4the centrifugal 10min of rpm, supernatant moves into new pipe, adds 0.4mL Virahol, upset, centrifugal 5min.Rinse precipitation with 70% ethanolic soln, dry after centrifugal.Add 170uL1 × TE, concussion mixing take gDNA as template, with screening primer amplification transgenic fragment.
Whether PCR primer carries out 0.8% agarose gel electrophoresis, observe foreign transgenes and be integrated in genome.
4, the extraction of total serum IgE and RT-PCR
Illustrate by Trizol reagent and carry out Total RNAs extraction, illustrate that carrying out reverse transcription obtains cDNA by reverse transcription reagents.Analyze to screen its expression of primer pair.
5, specific microRNA content in transgenic mouse milk is detected: be separated milk vesica, extract wherein RNA, reverse transcription is carried out through special reverse transcriptase primer, Real-time PCR detects wherein let-7g content, the results are shown in Figure 2, as shown in Figure 2, in transgenic (TG) mammary gland of mouse tissue let-7g content apparently higher than let-7g content in wild-type (WT) mammary gland of mouse tissue.
6, milk microRNA physiological function detects: with the milk of above-mentioned female transgenic positive mice nurture filial generation wild-type B6 mouse, control group adopts the milk nurture filial generation wild-type B6 mouse of female wild type B6 mouse, label is carried out to filial generation wild-type B6 mouse, spontaneous latter 7th day, the next day take each Mouse Weight, carry out the statistics of body weight gain situation, the results are shown in Figure 3.As shown in Figure 3, compare with control group, the generation mice body weight increase of let-7g gene overexpression to lactation has restraining effect (n=6).
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and can carry out many changes in the spirit and scope that the claims in the present invention limit to it, amendment, and even equivalence is changed, but all will fall within the scope of protection of the present invention.
Claims (4)
1. set up a kind of method of the fertilised non-human eggs for the functional study of milk microRNA, it is characterized in that: comprise the following steps:
(1) MMTV-let-7g plasmid is built: the flanking sequence choosing people let-7g sequence and each 150bp of upstream and downstream thereof on NCBI, the cloning primer of design containing EcoR I restriction enzyme site, take genome as template, through the above-mentioned sequence of pcr amplification, EcoR I is the single endonuclease digestion PCR primer obtained and the pBluescript II carrier containing mammary gland specific promoter MMTV-LTR respectively, CIP is to carrier dephosphorylation, after purifying, through T4 ligase enzyme, the two is connected, be transformed into competent cell DH5 α after connecting product order-checking correctly, set up recombinant plasmid MMTV-let-7g;
(2) zygote of mammary gland special process LAN let-7g transgenic mice is set up: with the above-mentioned recombinant plasmid of Spe I single endonuclease digestion, running gel reclaims the microinjection transgenosis component obtaining purifying, this component comprises mammary gland specific promoter MMTV-LTR, Let-7g and transcription termination signal; The transgenosis component of acquisition purifying is imported through microinjection in the zygote survived.
2. method according to claim 1, it is characterized in that: the carrier 3h of EcoR the I PCR primer that obtains of single endonuclease digestion and the pBluescript II containing mammary gland specific promoter MMTV-LTR respectively during described structure MMTV-let-7g plasmid, CIP is to carrier dephosphorylation 1h, 5 μ g products are obtained after purifying, through T4 ligase enzyme, the two is connected, competent cell DH5 α 50 μ L is transformed into after connecting product order-checking correctly, coated plate, next day, picking 5 bacterium colonies carry out order-checking qualification, determine recombinant plasmid MMTV-let-7g.
3. method according to claim 1, is characterized in that: the described cloning primer containing EcoR I restriction enzyme site is: 7g-CL-F:aatgagaattctccaaatgtggtg, 7g-CL-R:ccttgaattcatcttcaaacttcttg.
4. method according to claim 1, is characterized in that: the concentration of the transgenosis component described in adjustment is used for microinjection to 1ng ~ 2ng/uL.
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| CN111979270A (en) * | 2020-07-28 | 2020-11-24 | 扬州大学 | Construction method and application of mouse mammary tissue miRNA over-expression model |
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