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Transgene carrier for changing mouse movement ability and metabolic function by specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in skeletal muscle

A transgenic carrier and skeletal muscle-specific technology, applied in the field of genetic engineering, can solve problems such as unclear metabolism

Inactive Publication Date: 2012-12-19
CHINA PHARM UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, its metabolic role in these tissues remains unclear

Method used

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  • Transgene carrier for changing mouse movement ability and metabolic function by specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in skeletal muscle
  • Transgene carrier for changing mouse movement ability and metabolic function by specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in skeletal muscle
  • Transgene carrier for changing mouse movement ability and metabolic function by specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in skeletal muscle

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1: Inserting a DNA fragment of the mouse PEPCK-C coding region to construct pst228.

[0015] The vector pst215 in our laboratory was double-digested with BsaBI and AscI. The enzyme digestion system was 2 μg plasmid DNA, 3 μl 10× buffer, 1.5 μl BsaBI enzyme, 1.5 μl AscI enzyme, and ddH 2 O to make up the volume to 30 μl and incubate at 37°C for 3 hours. Then the digestion system was separated by electrophoresis in 1% agarose gel, and the 6.3kb DNA band was cut out, and the DNA was purified with a gel recovery purification kit. The specific steps are as follows: put the target DNA band excised under ultraviolet light into a 1.5ml centrifuge tube, add 3 times the volume of sol solution after weighing, and incubate at 55°C for 10 minutes to completely melt the agarose gel; After the temperature has dropped to room temperature, transfer the mixture to the attached column, centrifuge at 10,000g for 1 minute, and discard the effluent; add 650 μl of washing buffer, cen...

Embodiment 2

[0020] Example 2: Insert the promoter DNA fragment of mouse α-skeletal muscle actin gene to construct pst229.

[0021] Digest pst228 with Nrul and BsaBI, the enzyme digestion system is 2 μg plasmid DNA, 3 μl 10× buffer, 1.5 μl Nrul enzyme, 1.5 μl BsaBI enzyme, add ddH 2 O to make up the volume to 30 μl and incubate at 37°C for 3 hours. Then the digestion system was separated by electrophoresis in 1% agarose gel, and the DNA band with a size of 7.0 kb was cut out, and the DNA was purified with a gel recovery and purification kit. The specific steps are the same as above.

[0022] The promoter DNA fragment of the mouse α-skeletal muscle actin gene of 3.3 kb was obtained by PCR amplification, and the PCR conditions were as follows: 1 μ l (about 100 ng) mouse genomic DNA, primer 1 (TCG CGA ACG CGT GCCTTCCCTGTC) and primer 2 ( The final concentration of GAT CAC GAT CTA GTT TCT GCA AAG ACA AG) is 0.5 μM, the final concentration of dNTPs is 0.2 mM, 5 μl 10× buffer, 1 μl HiFi Taq en...

Embodiment 3

[0028] Example 3: Construction and identification of transgenic mice using the method of pronuclear injection.

[0029] The constructed pst229 transgenic vector was directly injected into the fertilized eggs to integrate the foreign gene into the DNA, and then the fertilized eggs were implanted into surrogate mother mice aged 4-5 weeks.

[0030] PEPCK-C mus After the transgenic mice were successfully bred, related studies on exercise and metabolism were carried out. The study found that compared with wild-type mice of the same age, PEPCK-C mus The transgenic mice had stronger exercise ability (mouse exercise treadmill test 158.25±54.59vs.2366±687.77, unit: m, P<0.01), and higher insulin sensitivity (area under the blood glucose-time curve of insulin tolerance test 16.27±1.16vs .15.19±0.90, unit: mmol / L, P<0.05), lower blood lipid level (1.64±0.20vs.5.19±2.02, unit: nmol / L, P<0.01), fasting blood glucose (7.73±0.35vs.6.48 ±0.78, unit: mmol / L), body weight (25.95±0.07vs.28.25±...

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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 [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a transgenic carrier for specifically expressing PEPCK-C in skeletal muscle to change the exercise capacity and metabolic function of mice. Background technique [0002] Phosphoenolpyruvate carboxykinase (PEPCK) is a rate-limiting enzyme that catalyzes the reaction of gluconeogenesis, that is, the reaction of converting oxaloacetate and GTP into phosphoenolpyruvate, GDP and CO2 . Currently, there are two known isoenzymes of PEPCK, namely PEPCK-M (mitochondrial form) and PEPCK-C (cytosolic form). PEPCK-C (also called PCK1) plays an important role in gluconeogenesis, and its research is also important More. At present, PEPCK-C has become a clear marker of liver gluconeogenesis, and the transcription level of this gene in the liver has become an important indicator for clinical evaluation of type 2 diabetes, that is, if the mRNA expression of PEPCK-C increa...

Claims

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Application Information

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IPC IPC(8): C12N15/85
Inventor 戴一凡冯玮任媛媛刘乃丰
Owner CHINA PHARM UNIV
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