CN105779495A - Method for realizing RNA (ribonucleic acid) overexpression by ovarian orthotopic injection and application of method - Google Patents
Method for realizing RNA (ribonucleic acid) overexpression by ovarian orthotopic injection and application of method Download PDFInfo
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Abstract
The invention belongs to the technical field of biology and discloses a method for realizing RNA (ribonucleic acid) overexpression by ovarian orthotopic injection and application of the method.RNA overexpression in a mouse ovary is realized by means of in-vivo transfection so as to provide a technical support for further researches on functions of RNA in the ovary. The method has the advantages that the defect of lack of targeting property of caudal vein over-expressed genes is avoided; transfection reagent consumption is reduced by dozens of times as compared with that of caudal vein injection, so that experiment cost is saved; excellent overexpression effects are achieved, and RNA expression level is raised remarkably as compared with that of a blank control group. The method which is minimally invasive has no influences on survival of mice.
Description
Technical field
The invention belongs to biological technical field, realize method and the application of RNA process LAN particularly to a kind of ovary in-situ injection.
Background technology
So far do not find by report same or similar with the inventive method.
Summary of the invention
For overcoming shortcoming and deficiency, a kind of method that the primary and foremost purpose of the present invention is in that to provide ovary in-situ injection to realize RNA process LAN of prior art.The present invention realizes RNA process LAN in mouse ovarian by the method for internal transfection, provides technical support for studying RNA function in ovary further.
Another object of the present invention is to the application that the above-mentioned ovary in-situ injection of offer realizes the method for RNA process LAN.
The purpose of the present invention is achieved through the following technical solutions: a kind of method that ovary in-situ injection realizes RNA process LAN, and operating procedure is as follows:
(1) transfection composite preparation, including lncRNA recombinant vector diluent and transfection reagent diluent:
Described lncRNA recombinant vector diluent:
The lncRNA recombinant vector of 1ug/ul: 6.25ul；
10% glucose solution 3.125 μ l；
Water 3.125 μ l；
Described transfection reagent diluent:
Transfection reagent 6.25 μ l；
10% glucose solution 6.25 μ l；
(2) internal transfection experiment operating procedure:
A. mouse anesthesia and Baoding: operation consent mice fasting 12 hours, draws the pentobarbital sodium of 0.3ml1% with 1ml syringe, and lumbar injection, in Mice Body, then on its Baoding to the plank being equipped with four spikes, will stretch extremity and is bonded on spike；
B. with razor to mouse web portion shaving, surgical field of view is exposed；
C. under aseptic condition, cut skin 2cm with pubis leading edge midpoint to tailing edge abdomen diapire median line at umbilicus, cut hunter's line and peritoneum, open abdominal cavity；Find body of uterus right uterine angle with ophthalmic tweezers at suitable order, find forward right ovary along cornua uteri, fix ovary with ophthalmic tweezers gently；
D. the Bohemian glass capillary tube that diameter is 1mm being rotated calcination on flame so that it is deliquescing, rapid trombone slide fractures from centre so that it is internal diameter becomes 200 μm；Being subsequently attached on mouthful suction pipe, ovary 9～11mm is gently inserted after drawing 25 μ l transfection composites in capillary tube one end, the transfection composite in step (1) is blown into ovary slowly, as experimental group；
E. find left ovary by same method, the transfection composite containing skeleton carrier is blown into ovary slowly, as experiment contrast group；
F. careful original anatomical position of also being received back by ovary, utilizes absorbable suture suture muscles layer and skin respectively, at operative incision suture iodophor disinfection after stitching, for three days on end；
G. being incubated with heat-insulating lamp postoperative daytime, night closes, and recovers mice normal diet；
H. process LAN effect detection；After transfecting 5 days, win both sides ovary respectively；Cutting the sub-fraction of both sides ovary under stereomicroscope respectively, extract total tissue RNA, reverse transcription becomes cDNA, utilizes qPCR to detect the process LAN level of lncRNA.
Transfection reagent described in step (1) be Ying Geen bio tech ltd, Beijing EntransterTM-invivo body in transfection reagent.
Mouth suction pipe described in step (2) is silica gel hose.
LncRNA recombinant vector described in step (1) is obtained by following method:
1) .PCR design of primers: the cDNA sequence for mouse ovarian lncRNA designs PCR primer；
2) .PCR template DNA: extract the total serum IgE of C57BL/6 Mouse Ovary Tissues and obtain the cDNA template for PCR by reverse transcription；Use TaKaRaHSDNAPolymerase enzymatic amplification；PCR reaction takes amplified production 3 μ l and is separately added into 1 μ l5 × DNAloadingbuffer 120V electrophoresis in 1% agarose gel after terminating, and sees whether bright band under uviol lamp, prepares the purification of PCR primer；
3) .PCR amplified production purification: use TaKaRaMiniBESTAgaroseGelDNAExtractionKitVer.4.0 gel to reclaim test kit and carry out the purification of pcr amplification product；
4) pcr amplification product and MouseZP3promoter (JD#445 skeleton carrier) skeleton carrier endonuclease reaction: pcr amplification product and MOUSEZP3PROMOTER (JD#445 skeleton carrier) skeleton carrier are carried out double digestion respectively with NEBXho I and SmaI restricted enzyme, digestion products is electrophoresis observation under uviol lamp in the agarose gel of 1%, uses TaKaRaMiniBESTAgaroseGelDNAExtractionKitVer.4.0 gel to reclaim test kit and carry out digestion products recovery after cutting target DNA band；
5). digestion products connects and converts: utilize DNALigationKitVer.2.1 that genes of interest is connected to skeleton carrier, take 10 μ l to connect products and proceed to and be placed in the DH5 α of 5 minutes of thawing on ice, on 100 μ l bacterium solution even spread after conversion to the LB solid medium plate containing ammonia benzyl resistance, 37 DEG C of constant incubators will be inverted overnight incubation；
6). picking monoclonal bacterium colony is cultivated and extracts recombinant plasmid dna: with in the antibiotic 5mlLB fluid medium of monoclonal colony inoculation benzyl containing ammonia on sterilizing toothpick picking flat board, 37 DEG C of shaking table overnight incubation, extract plasmid DNA next day, use the little extraction reagent kit of high-purity plasmid of Tian Gen biochemical technology company limited to carry out the extraction of plasmid DNA；Obtain lncRNA recombinant vector.
Above-mentioned ovary in-situ injection realizes the application in ovary is studied of the method for RNA process LAN.
The present invention has such advantages as relative to prior art and effect:
1. avoid the tail vein process LAN gene shortcoming without targeting；
2. the amount ratio tail vein injection of transfection reagent decreases tens times, has saved experimental cost；
3. process LAN effect is fine, and compared with blank group, rna expression level significantly raises.
4. the method for the Wicresoft that the present invention uses, does not affect mouse survival.
Accompanying drawing explanation
Fig. 1 is the expression testing result figure in lncRNA-NONMMUT047274 body after transfected ovary.* P < 0.01, difference is extremely notable.
Detailed description of the invention
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Embodiment 1
1) .PCR design of primers.CDNA sequence for mouse ovarian lncRNA-NONMMUT047274 designs PCR primer, and primer sequence is as follows:
2) .PCR template DNA.Extract the total serum IgE of C57BL/6 Mouse Ovary Tissues and obtain the cDNA template for PCR by reverse transcription.Use TaKaRaHSDNAPolymerase enzymatic amplification.PCR reaction takes amplified production 3 μ l and is separately added into 1 μ l5 × DNAloadingbuffer 120V electrophoresis in 1% agarose gel after terminating, and sees whether bright band under uviol lamp, prepares the purification of PCR primer.
3) .PCR amplified production purification.Use TaKaRaMiniBESTAgaroseGelDNAExtractionKitVer.4.0 gel to reclaim test kit and carry out the purification of pcr amplification product.
4) .PCR amplified production and MOUSEZP3PROMOTER (JD#445 skeleton carrier) skeleton carrier endonuclease reaction.With NEBXho I and SmaI restricted enzyme, pcr amplification product and MOUSEZP3PROMOTER (JD#445 skeleton carrier) skeleton carrier are carried out double digestion respectively, digestion products is electrophoresis observation under uviol lamp in the agarose gel of 1%, uses TaKaRaMiniBESTAgaroseGelDNAExtractionKitVer.4.0 gel to reclaim test kit and carry out digestion products recovery after cutting target DNA band.
5). digestion products connects and converts.Utilize DNALigationKitVer.2.1 that genes of interest is connected to skeleton carrier, take 10 μ l to connect products and proceed to and be placed in the DH5 α of 5 minutes of thawing on ice, on 100 μ l bacterium solution even spread after conversion to the LB solid medium plate containing ammonia benzyl resistance, 37 DEG C of constant incubators will be inverted overnight incubation.
6). picking monoclonal bacterium colony is cultivated and extracts recombinant plasmid dna.With in the antibiotic 5mlLB fluid medium of monoclonal colony inoculation benzyl containing ammonia on sterilizing toothpick picking flat board, 37 DEG C of shaking table overnight incubation, next day extracts plasmid DNA.The little extraction reagent kit of high-purity plasmid (centrifugal column type) using TIANGEN Biotech (Beijing) Co., Ltd. carries out the extraction of plasmid DNA.
7). whether sequence verification lncRNA has inserted MOUSEZP3PROMOTER (JD#445 skeleton carrier) skeleton carrier.The recombiant plasmid of extraction send work biological engineering (Shanghai) limited company (raw work) check order.
8) process LAN experiment in .lncRNA recombinant vector body.Transfection reagent in the EntransterTM-invivo body of Ying Geen bio tech ltd, Beijing is used to carry out process LAN experiment in lncRNA recombinant vector body.Preparation and the specific experiment step of transfection reagent are as follows:
1. transfection cocktail preparation.See following table:
2. internal transfection experiment operating procedure:
A. mouse anesthesia and Baoding: operation consent mice fasting 12 hours, the pentobarbital sodium of 0.3ml1% is drawn with new 1ml syringe, lumbar injection, in Mice Body, then on its Baoding to the plank (30cm × 20cm) being equipped with four spikes, will stretch extremity and is bonded on spike.
B. with razor to mouse web portion shaving, surgical field of view is exposed.
C. under aseptic condition, cut skin 2cm with pubis leading edge midpoint to tailing edge abdomen diapire median line at umbilicus, cut hunter's line and peritoneum, open abdominal cavity；Find body of uterus right uterine angle with ophthalmic tweezers at suitable order, find forward right ovary along cornua uteri, fix ovary with ophthalmic tweezers gently.
D. the Bohemian glass capillary tube that diameter is 1mm being rotated calcination on flame so that it is deliquescing, rapid trombone slide fractures from centre so that it is internal diameter becomes 200 μm；Being subsequently attached on a mouthful suction pipe (silica gel hose), capillary tube one end is gently inserted ovary after drawing 25 μ l transfection composites and is about 10mm, transfection composite (recombinant plasmid dna) is blown into ovary slowly, as experimental group.
E. find left ovary by same method, transfection composite (ZP3 skeleton carrier) is blown into ovary slowly, as experimental group matched group.
F. careful original anatomical position of also being received back by ovary, utilizes absorbable suture suture muscles layer and skin respectively, at operative incision suture iodophor disinfection after stitching, for three days on end.
G. being incubated with heat-insulating lamp postoperative daytime, night closes, and recovers mice normal diet.
9). process LAN effect detection.After transfecting 5 days, win both sides ovary respectively.Cutting the sub-fraction of both sides ovary under stereomicroscope respectively, extract total tissue RNA, reverse transcription becomes cDNA, utilizes qPCR to detect the process LAN level of lncRNA.Testing result as it is shown in figure 1, after transfection the expression of experimental group lncRNA-NONMMUT047274 increase about 125 times.
Above-described embodiment is the present invention preferably embodiment; but embodiments of the present invention are also not restricted to the described embodiments; the change made under other any spirit without departing from the present invention and principle, modification, replacement, combination, simplification; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (5)
1. the method that an ovary in-situ injection realizes RNA process LAN, it is characterised in that: its operating procedure is as follows:
(1) transfection composite preparation, including lncRNA recombinant vector diluent and transfection reagent diluent:
Described lncRNA recombinant vector diluent:
The lncRNA recombinant vector of 1ug/ul: 6.25ul；
10% glucose solution 3.125 μ l；
Water 3.125 μ l；
Described transfection reagent diluent:
Transfection reagent 6.25 μ l；
10% glucose solution 6.25 μ l；
(2) internal transfection experiment operating procedure:
A. mouse anesthesia and Baoding: operation consent mice fasting 12 hours, draws the pentobarbital sodium of 0.3ml1% with 1ml syringe, and lumbar injection, in Mice Body, then on its Baoding to the plank being equipped with four spikes, will stretch extremity and is bonded on spike；
B. with razor to mouse web portion shaving, surgical field of view is exposed；
C. under aseptic condition, cut skin 2cm with pubis leading edge midpoint to tailing edge abdomen diapire median line at umbilicus, cut hunter's line and peritoneum, open abdominal cavity；Find body of uterus right uterine angle with ophthalmic tweezers at suitable order, find forward right ovary along cornua uteri, fix ovary with ophthalmic tweezers gently；
D. the Bohemian glass capillary tube that diameter is 1mm being rotated calcination on flame so that it is deliquescing, rapid trombone slide fractures from centre so that it is internal diameter becomes 200 μm；Being subsequently attached on mouthful suction pipe, ovary 9～11mm is gently inserted after drawing 25 μ l transfection composites in capillary tube one end, the transfection composite in step (1) is blown into ovary slowly, as experimental group；
E. find left ovary by same method, the transfection composite containing skeleton carrier is blown into ovary slowly, as experiment contrast group；
F. careful original anatomical position of also being received back by ovary, utilizes absorbable suture suture muscles layer and skin respectively, at operative incision suture iodophor disinfection after stitching, for three days on end；
G. being incubated with heat-insulating lamp postoperative daytime, night closes, and recovers mice normal diet；
H. process LAN effect detection；After transfecting 5 days, win both sides ovary respectively；Cutting the sub-fraction of both sides ovary under stereomicroscope respectively, extract total tissue RNA, reverse transcription becomes cDNA, utilizes qPCR to detect the process LAN level of lncRNA.
2. the method that ovary in-situ injection according to claim 1 realizes RNA process LAN, it is characterised in that: the transfection reagent described in step (1) be Ying Geen bio tech ltd, Beijing EntransterTM-invivo body in transfection reagent.
3. the method that ovary in-situ injection according to claim 1 realizes RNA process LAN, it is characterised in that: the mouth suction pipe described in step (2) is silica gel hose.
4. the method that ovary in-situ injection according to claim 1 realizes RNA process LAN, it is characterised in that: the lncRNA recombinant vector described in step (1) is obtained by following method:
1) .PCR design of primers: the cDNA sequence for mouse ovarian lncRNA designs PCR primer；
2) .PCR template DNA: extract the total serum IgE of C57BL/6 Mouse Ovary Tissues and obtain the cDNA template for PCR by reverse transcription；Use TaKaRaHSDNAPolymerase enzymatic amplification；PCR reaction takes amplified production 3 μ l and is separately added into 1 μ l5 × DNAloadingbuffer 120V electrophoresis in 1% agarose gel after terminating, and sees whether bright band under uviol lamp, prepares the purification of PCR primer；
3) .PCR amplified production purification: use TaKaRaMiniBESTAgaroseGelDNAExtractionKitVer.4.0 gel to reclaim test kit and carry out the purification of pcr amplification product；
4) .PCR amplified production and MouseZP3promoter skeleton carrier endonuclease reaction: pcr amplification product and MouseZP3promoter skeleton carrier are carried out double digestion respectively with NEBXho I and SmaI restricted enzyme, digestion products is electrophoresis observation under uviol lamp in the agarose gel of 1%, uses TaKaRaMiniBESTAgaroseGelDNAExtractionKitVer.4.0 gel to reclaim test kit and carry out digestion products recovery after cutting target DNA band；
5). digestion products connects and converts: utilize DNALigationKitVer.2.1 that genes of interest is connected to skeleton carrier, take 10 μ l to connect products and proceed to and be placed in the DH5 α of 5 minutes of thawing on ice, on 100 μ l bacterium solution even spread after conversion to the LB solid medium plate containing ammonia benzyl resistance, 37 DEG C of constant incubators will be inverted overnight incubation；
6). picking monoclonal bacterium colony is cultivated and extracts recombinant plasmid dna: with in the antibiotic 5mlLB fluid medium of monoclonal colony inoculation benzyl containing ammonia on sterilizing toothpick picking flat board, 37 DEG C of shaking table overnight incubation, extract plasmid DNA next day, use the little extraction reagent kit of high-purity plasmid of Tian Gen biochemical technology company limited to carry out the extraction of plasmid DNA；Obtain lncRNA recombinant vector.
5. the ovary in-situ injection described in any one of Claims 1 to 4 realizes the application in ovary is studied of the method for RNA process LAN.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110384800A (en) * | 2019-07-19 | 2019-10-29 | 广东省实验动物监测所 | Application of the LncRNA XLOC_075168 in the drug that preparation promotes angiogenesis |
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Non-Patent Citations (3)
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| ADMIN: "EntransterTM-in vivo体内转染试剂", 《英格恩技术的博客：HTTPS://WWW.ENGREEN.CN/2015/08/ENTRANSTERTM-IN-VIVO-TRANSFECTION-INIVO/》 * |
| 张文辉等: "长链非编码RNA作为潜在药物靶点的研究进展", 《药学进展》 * |
| 张旭: "LncRNA h19在非小细胞肺癌中的表达及对细胞的生物学行为的影响", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110384800A (en) * | 2019-07-19 | 2019-10-29 | 广东省实验动物监测所 | Application of the LncRNA XLOC_075168 in the drug that preparation promotes angiogenesis |
| CN110384800B (en) * | 2019-07-19 | 2021-06-08 | 广东省实验动物监测所 | Application of LncRNA XLOC _075168 in preparation of medicine for promoting angiogenesis |
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Application publication date: 20160720 |