CN102827874A - Transgene carrier for changing mouse movement ability and metabolic function by specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in skeletal muscle - Google Patents
Transgene carrier for changing mouse movement ability and metabolic function by specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in skeletal muscle Download PDFInfo
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- CN102827874A CN102827874A CN2012103455508A CN201210345550A CN102827874A CN 102827874 A CN102827874 A CN 102827874A CN 2012103455508 A CN2012103455508 A CN 2012103455508A CN 201210345550 A CN201210345550 A CN 201210345550A CN 102827874 A CN102827874 A CN 102827874A
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Abstract
The invention discloses a transgene carrier for changing the mouse movement ability and the metabolic function by the specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in the muscular tissue. The transgene carrier comprises the promoter of the gene of the 3.3kb alpha-skeletal actin of a mouse, the cDNA (deoxyribonucleic acid) of the PEPCK of the mouse and the 3'-end no-translation area of a bovine growth hormone gene (bGH); the transgene carrier is provided with a resistance gene-puromycin gene used for screening in mammalian cells, and a resistance gene-ampicillin gene used for screening in prokaryotic cells. The transgene carrier disclosed by the invention can be used for cloning a somatic cell so as to construct a PEPCK-Cmus transgene mouse.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to the specific expressed PEPCK-C of a kind of Skelettmuskel to change the transgene carrier of mouse movement ability and metabolic function.
Background technology
PCK (phosphoenolpyruvate carboxykinase; PEPCK) be a rate-limiting enzyme of catalysis glyconeogenesis (gluconeogenesis) reaction, promptly change the reaction of PEP, GDP and CO2 into by oxaloacetic acid and GTP.The present known two kinds of PEPCK isozymes that have are respectively PEPCK-M (mitochondrial form) and PEPCK-C (cytosolic form), and PEPCK-C (also being PCK1) plays an important role in glyconeogenesis, and is also more to its research.At present PEPCK-C has become a clear and definite affinity tag of liver glyconeogenesis, and this gene transcription level becomes the important indicator that diabetes B is clinically estimated in the liver, if promptly the mRNA of PEPCK-C expresses and increases, the speed of glyconeogenesis just is bound to increase.The PEPCK-C genes encoding proteolytic enzyme of a 63kDa, mainly in liver and renal cortex, express, and catalysis glyconeogenesis reaction; In liver and white and BAT, PEPCK-C also participates in glycerine heteroplasia reaction (glyceroneogenesis); In addition, PEPCK-C works in the reaction (cataplerosis) of running off, to remove the negatively charged ion in the tricarboxylic acid cycle; The PEPCK-C enzyme also extensively is present in the mammiferous tissue simultaneously, comprises small intestine, colon, mammary gland, suprarenal gland, lung and muscle.But its metabolism in these tissues is not clear and definite yet.
The activity of PEPCK-C in different tissues regulated and control on transcriptional level.For example; The promoter sequence that specifically expressing PEPCK-C gene only need be from-460bp to+69bp in liver; PEPCK-C gene transcription level that nutritive substance and hormone are adjustable; CAMP, glucocorticosteroid and Regular Insulin can both be regulated the PEPCK-C gene transcription, and have confirmed their corresponding regulating and controlling sequences on the PEPCK-C promotor.
Because the major function of PEPCK-C in different tissues is different,, different tissue-specific gene knockouts or mistake cause multiple different phenotype so expressing.Under the normal circumstances, PEPCK-C in Skelettmuskel, do not express or expression amount very low.PEPCK-C comes the catalysis pyruvic acid to change a glucose into the energy of 6 ATP, and the anaerobic glycolysis per minute in Skelettmuskel is separated a glucose, but can only generate 2 ATP.We are expected at over-expresses PEPCK-C in the Skelettmuskel, and triglyceride in blood and free fatty acids possibly all can transport to Skelettmuskel, and more energy is provided, and possibly influence the metaboilic level of body.
Summary of the invention
Technical problem to be solved by this invention provide a kind of can be in Skelettmuskel specific expressed PEPCK-C to change the transgene carrier of mouse movement ability and metabolic function.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is following:
A kind of can be in Skelettmuskel the transgenic fragment of specific expressed PEPCK-C comprise 3 ' end untranslated region territory of α-Skelettmuskel Actin muscle (α-skeletal actin) promotor of gene, the cDNA of mouse PEPCK-C and the bovine growth hormone gene (bGH) of mouse, and screen resistant gene-penbritin (ampicillin) gene (see figure 2) of usefulness in resistant gene-tetracycline (Puromycin) gene that has screening usefulness in the mammalian cell and the prokaryotic cell prokaryocyte.
3.3kb mouse α-Skelettmuskel Actin muscle (α-skeletal actin) promotor can guarantee that the gene in downstream is specific expressed in Skelettmuskel.
The complete sequence of the transgene carrier of the Skelettmuskel specifically expressing mouse PEPCK-C that the present invention makes up is shown in SEQ ID No:1; Wherein the 228-3525 base is the promoter region of α-Skelettmuskel Actin muscle (α-skeletal actin) gene; The 3604-5472 base is mouse PEPCK-C coding region; The 6386-6610 base is bGH a 3 ' end untranslated region; The 6693-7292 base is tetracycline (Puromycin) resistant gene, and the 9337-10336 base is penbritin (Ampicillin) resistant gene.
Transgene carrier of the present invention can be used for somatic cell clone, to make up the PEPCK-C transgenic mice that can change mouse movement ability and metabolic function.
Description of drawings
Fig. 1 is a techniqueflow chart of the present invention.
Fig. 2 is the synoptic diagram of the pST229 transgene carrier that makes up of the present invention.
Embodiment
According to following embodiment, can understand the present invention better.Yet, those skilled in the art will readily understand that the described content of embodiment only is used to explain the present invention, and the present invention that should also can not limit in claims to be described in detail.
Embodiment 1: insert mouse PEPCK-C coding region dna fragmentation, make up pst228.
With BsaBI and AscI double digestion, the enzyme system of cutting is 2 μ g DNAs with this breadboard carrier pst215,3 μ l10 * buffer, and 1.5 μ lBsaBI enzymes, 1.5 μ lAscI enzymes add ddH
2O supplies volume to 30 μ l, and 37 ° of C temperature were bathed 3 hours.Then the enzyme system of cutting is passed through electrophoretic separation in 1% sepharose, cut out the DNA band of 6.3kb size, reclaim the purification kit purify DNA with glue.Concrete steps do, UV-light is downcut the target DNA band down pack in the 1.5ml centrifuge tube, and the back of weighing adds the sol solutions of 3 times of volumes, and 55 ℃ of insulations 10 minutes are melted sepharose fully; After the gel that melts treats that temperature is reduced to room temperature, mixed solution is changed in the attached column, 10, centrifugal 1 minute of 000g outwells effluent; Add 650 μ l rinsing damping fluids, 10, centrifugal 1 minute of 000g outwells effluent; The adsorption column recentrifuge, 10,000g is centrifugal, 1 minute, outwells effluent; Adsorption column is transferred in the new 1.5ml centrifuge tube, added 50 μ lddH
2O placed 1 minute, and 10,000g collected purified product in centrifugal 1 minute.
Synthetic (Invitrogen) obtains mouse PEPCK-C coding region dna fragmentation through full gene, and with BsaBI and AscI double digestion, the enzyme system of cutting is 3 μ g DNAs, 3 μ l10 * buffer, and 1.5 μ lBsaBI enzymes, 1.5 μ lAscI enzymes add ddH
2O supplies volume to 30 μ l, and 37 ° of C temperature were bathed 3 hours.Then the enzyme system of cutting is passed through electrophoretic separation in 1% sepharose, cut out the DNA band of 1.9kb size, reclaim the purification kit purify DNA with glue.。
Pst215 (BsaBI/AscI) is connected with mouse PEPCK-C coding region dna fragmentation (BsaBI/AscI), adds 2 μ l carriers in the ligation, 6 μ l insert fragment, 1 μ lT4DNA ligase enzyme, and 16 ° of C temperature were bathed 16 hours.
Be transformed in the competent cell of E.coli connecting product.The Trans5 α that gets a pipe 50 μ l is placed on ice by the attitude cell, treat that it melts after, to wherein adding the above-mentioned connection product of 4 μ l, flick mixing, the back is in hatching 30 minutes on ice; 42 ℃ of thermal shocks 30 seconds, after left standstill on ice 10 minutes; Add the nonresistant LB liquid nutrient medium of 500 μ l, 37 ℃ of concussions were cultivated 1 hour; 4, centrifugal 5 minutes of 000rpm siphons away, and keeps the substratum about 100 μ l, is coated on the LB culture medium flat plate that contains amicillin resistance after with bacterial precipitation piping and druming evenly with pipettor.Place 37 ℃ of incubators to cultivate 16 hours flat board then.
After clonal growth comes out; Choose the mono-clonal bacterium colony and contain to 3ml in the LB nutrient solution of penbritin, 37 ℃ of concussions were cultivated 12 hours, extracted DNA; Cut evaluation with the EcoRI enzyme, can obtain four DNA bands of 4.0kb, 2.7kb, 1.9kb, 0.5kb in the positive colony.Further confirm the exactness of positive colony again through order-checking, the clone who obtains is pst228.
Embodiment 2: insert the promoter dna fragment of mouse α-Skelettmuskel actin gene, make up pst229.
With Nrul and BsaBI double digestion, the enzyme system of cutting is 2 μ g DNAs with pst228,3 μ l10 * buffer, and 1.5 μ lNrul enzymes, 1.5 μ lBsaBI enzymes add ddH
2O supplies volume to 30 μ l, and 37 ° of C temperature were bathed 3 hours.Then the enzyme system of cutting is passed through electrophoretic separation in 1% sepharose, cut out the DNA band of 7.0kb size, reclaim the purification kit purify DNA with glue.Concrete steps are the same.
Obtain the promoter dna fragment of mouse α-Skelettmuskel actin gene of 3.3kb through pcr amplification; The PCR condition is following: 1 μ l (about 100ng) musculus cdna group DNA; The final concentration of primer 1 (TCG CGA ACG CGT GCCTTCCCTGTC) and primer 2 (GAT CAC GAT CTA GTT TCT GCA AAG ACA AG) is 0.5 μ M, and the final concentration of dNTPs is 0.2mM, 5 μ l10 * buffer; 1 μ lHiFi Taq enzyme adds ddH
2O supplies volume to 50 μ l.PCR circulation and is 95 ° of C, 2 minutes; Then be 95 ° of C, 30 seconds, 55 ° of C, 60 seconds, 72 ° of C, 3 minutes, 35 circulations; 72 ° of C, 5 minutes; 4 ° of C, insulation.The PCR product is passed through electrophoretic separation in 1% sepharose, cut out the DNA band of 3.3kb size, reclaim the purification kit purify DNA with glue.Concrete steps are the same.
The promoter dna fragment of mouse α-Skelettmuskel actin gene of the 3.3kb that pcr amplification is obtained is connected on the pEASY-T3 carrier (full formula King Company) through the TA clone.1 μ lpEASY-T3 adds the promoter dna fragment of mouse α-Skelettmuskel actin gene of 4 μ l3.3kb, 25 ° of C, 30 minutes.Then ligation is transformed in the E.coli competent cell, is applied to the cell after transforming on the LB flat board that contains IPTG and X-Gal and penbritin and grows.After clonal growth came out, the mono-clonal bacterium colony of choosing white contained to 3ml in the LB nutrient solution of penbritin, and 37 ℃ of concussions were cultivated 12 hours, extracted DNA, cut evaluation with the EcoRI enzyme, can obtain two DNA bands of 3.0kb and 3.3kb in the positive colony.Further confirm the exactness of positive colony again through order-checking, the clone who obtains is pPEK.
From pPEK with Nrul and BsaBI double digestion to cut out the promoter DNA of mouse α-Skelettmuskel actin gene, the enzyme system of cutting is 3 μ gpPEK3DNA, 3 μ l10 * buffer, 1.5 μ lNrul enzymes, 1.5 μ lBsaBI enzymes add ddH
2O supplies volume to 30 μ l, and 37 ° of C temperature were bathed 3 hours.Then the enzyme system of cutting is passed through electrophoretic separation in 1% sepharose, cut out the DNA band of 3.3kb size, reclaim the purification kit purify DNA with glue.Concrete steps are the same.
The promoter DNA (Nrul/BsaBI) of pst228 (Nrul/BsaBI) and mouse α-Skelettmuskel actin gene is connected, and ligation is the same.
Be transformed in the competent cell of E.coli connecting product.Concrete steps are the same.
After clonal growth comes out; Choose the mono-clonal bacterium colony and contain to 3ml in the LB nutrient solution of penbritin, 37 ℃ of concussions were cultivated 12 hours, extracted DNA; Cut evaluation with the EcoRI enzyme, can obtain four DNA bands of 6.1kb, 2.1kb, 1.4kb and 0.8kb in the positive colony.Further confirm the exactness of positive colony again through order-checking, the clone who obtains is pst229, i.e. the transgene carrier that the present invention describes.
Embodiment 3: utilize the method for procaryotic injection to make up transgenic mice and identify.
The pst229 transgene carrier that builds is directly injected zygote, make exogenous origin gene integrator in DNA, then zygote is implanted in the 4-5 female mouse body of replace-conceive in age in week.
PEPCK-C
MusBehind the transgenic mice reproductive success, move and the correlative study of metabolism aspect, discover, with wild-type mice of the same age relatively, PEPCK-C
MusTransgenic mice motor capacity stronger (the dull and stereotyped test of mouse movement 158.25 ± 54.59vs.2366 ± 687.77, unit: m, P<0.01), insulin sensitivity higher (area 16.27 ± 1.16vs.15.19 ± 0.90 under insulin tolerance test blood sugar-time curve, unit: mmol/>L, P<0.05), lipid level lower (1.64 ± 0.20vs.5.19 ± 2.02, unit: nmol/>L, P<0.01); Fasting plasma glucose (7.73 ± 0.35vs.6.48 ± 0.78; Unit: mmolL), body weight (25.95 ± 0.07vs.28.25 ± 0.35; Unit: g), the stomach fat ratio do not have difference (16.14 ± 1.92vs.17.52 ± 1.20, unit: %), but through in tissue slice H&E dyeing and the oil red coloration result discovery Skelettmuskel obvious fat accumulation is arranged.Presentation of results PEPCK-C gene is specific expressed in mice skeletal; Improved exercise tolerance and the insulin sensitivity of mouse and made blood fat maintain lower level, further specified the energy metabolism that the PEPCK-C gene expresses body and play an important role in Skelettmuskel.
Claims (2)
1. the specific expressed PEPCK-C of Skelettmuskel is to change the transgene carrier of mouse movement ability and metabolic function; It is characterized in that; Said transgene carrier contains cDNA and 3 ' the end untranslated region territory of bovine growth hormone gene of promotor, the mouse PEPCK-C of 3.3kb α-Skelettmuskel actin gene of mouse, and screens the resistant gene-penbritin gene of usefulness in the resistant gene-tetracycline gene that has screening usefulness in the mammalian cell and the prokaryotic cell prokaryocyte.
2. the specific expressed PEPCK-C of Skelettmuskel according to claim 1 is to change the transgene carrier of mouse movement ability and metabolic function, and its base complete sequence is shown in SEQ ID No.1.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103102A (en) * | 2017-05-09 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of skeletal muscle specificity PCK1 expression vectors virus-mediated AAV1 and application thereof |
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Non-Patent Citations (4)
| Title |
|---|
| 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 19930105 Brennan K J等 Quantitative analysis of the human alpha-skeletal actin gene in transgenic mice 第268卷, 第1期 * |
| BRENNAN K J等: "Quantitative analysis of the human α-skeletal actin gene in transgenic mice", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| HAKIMI P等: "Overexpression of the Cytosolic Form of Phosphoenolpyruvate Carboxykinase (GTP) in Skeletal Muscle Repatterns Energy Metabolism in the Mouse", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| YANG JQ等: "What is the metabolic role of phosphoenolpyruvate carboxykinase", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108103102A (en) * | 2017-05-09 | 2018-06-01 | 北京五加和分子医学研究所有限公司 | A kind of skeletal muscle specificity PCK1 expression vectors virus-mediated AAV1 and application thereof |
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Application publication date: 20121219 |