CN101921795A - Method for establishing porphyria cutanea tarda (PCT) transgenic mouse model - Google Patents
Method for establishing porphyria cutanea tarda (PCT) transgenic mouse model Download PDFInfo
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Abstract
The invention relates to the technical field of biology, in particular to a method for establishing a porphyria cutanea tarda (PCT) transgenic mouse model. The invention provides an animal model capable of stably inheriting and spontaneously generating a series of biochemical and pathologic characteristics conforming to the PCT. So far, the pathogeny and the pathogenesis of the PCT are not completely clear, and research reports about HO-1 participating in the PCT pathogenesis of human beings or animals are not seen. In the invention, a mouse model capable of spontaneously representing the PCT symptoms is established by overexpressing the HO-1 through a transgenic technology, thereby not only theoretically supplementing the pathogeny and the pathogenesis of the PCT, but also providing more valuable new ideas for the treatment of the PCT.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method of setting up porphyria cutanea tarda (PCT) transgenic mouse model.
Background technology
Porphyria cutanea tarda (porphyria cutanea tarda, PCT) be because the 5th enzyme---uroporphyrinogen decarboxylase (the uroporphyrinogen decarboxylase in the protoheme biosynthetic process, URO-D) molecular defect or activity reduce and a kind of modal porphyria of initiation, how the middle age with sequela, the male sex is more.The research of relevant PCT sees nineteen fifty the earliest, and in Turkey, people have caused the great outburst of a PCT because of having eaten the wheat seed that sprayed the mycocide Perchlorobenzene.Hereafter people bring out animal PCT with chlorating hydrocarbon polymers such as Perchlorobenzenes.Rodent is taken in iron, the synthetic precursor δ-An Ji-γ-Tong Wusuan of protoheme, alcohol etc. also can bring out PCT.The another kind of animal model that is used to study PCT is exactly the URO-D knock out mice.But heme oxidase-1 is not arranged as yet, and (Heme Oxygenase-1 HO-1) brings out the research report of human or animal PCT.(heme oxygenase, HO) the catalysis protoheme decomposes generation iron, carbon monoxide and uteroverdine to heme oxidase.The applicant is making after general crosses transgenosis C57BL/6/DBA2 (BDF1) mouse of expressing HO-1, finds that this transgenosis male mice suffered from porphyria cutanea tarda.This porphyria cutanea tarda animal model is expressed based on genetically modified mistake of HO-1.
Summary of the invention
Above-mentioned technical problem of the present invention is mainly solved by following technical proposals:
A kind of method of setting up porphyria cutanea tarda (PCT) transgenic mouse model is characterized in that, may further comprise the steps:
Step 2 is used micro-injection method the gene order that makes up is injected C57BL/6/DBA2 (BDF1) mouse fertilized egg;
Step 3 is implanted the zygote in the step 2 in the uterine tube of false pregnancy mouse;
Step 4 produces F0 for transgenic mice, is integrated with exogenous HO-1 expressed sequence in this mouse genome;
In above-mentioned a kind of method of setting up porphyria cutanea tarda (PCT) transgenic mouse model, the concrete orientation of described structure one linear transgenic sequence is: amplification HO-1 gene order in the mouse genome is cloned into the pCAGG carrier.HO-1 cDNA is started by chicken β-actin promotor and cmv enhancer, and HO-1 connects rabbit β-globin poly (A) termination signal, and plasmid is cut through SalI andDraI enzyme, obtains linear transgenic sequence.
Therefore, the present invention has following advantage: cross expression HO-1 by transgenic technology, set up the mouse model of the spontaneous reproduction porphyria cutanea tarda of a kind of energy symptom, not only done theoretic replenishing for the cause of disease of PCT, pathogenesis, more its treatment provides how valuable new concept.On the other hand, the present invention still has pathogenic producing evidence for proof HO-1 crosses expression, provides theory and experimental basis for utilizing HO-1 protection body from now on.
Description of drawings
Accompanying drawing 1 is a microinjection transgenosis member synoptic diagram;
Accompanying drawing 2 is HO-1 expression and HO determinations of activity in transgenosis (Tg) and wild-type (WT) mouse liver, and A detects the expression level synoptic diagram of HO-1 in Tg and WT mouse liver for Western blot; B is the active synoptic diagram of HO, and * compares p＜0.05 with brood wild-type mice;
Accompanying drawing 3 is that male HO-1 transgenosis (Tg) mouse suffers from PCT.Wherein, A and B are that the tooth of male HO-1Tg mouse sends red fluorescence (shown in the arrow) when being subjected to uviolizing; C is the uroporphyrin figure that tooth sends red fluorescence (erythrodontia mouse) and wild-type mice.URO, uroporphyrin; HEPTA, hepatacarboxyporphyrin; COPRO, coproporphyrin; D and E are the peculiar pin sample of PCT content (shown in the arrow) in the liver of erythrodontia mouse, * 400.。
Embodiment
Below by embodiment, and in conjunction with the accompanying drawings, technical scheme of the present invention is described in further detail.
Embodiment:
In conjunction with the accompanying drawings, following examples only are used to the present invention is described and do not limit the scope of the invention.
One, obtain goal gene:
The design primer, amplification HO-1 gene order is cloned into the pCAGG carrier in the mouse genome.HO-1 cDNA is started by chicken β-actin promotor and cmv enhancer, and HO-1 connects rabbit β-globin poly (A) termination signal (Fig. 1).Plasmid is cut through SalI and DraI enzyme, obtains linear transgenic sequence, and purifying reclaims and is used for microinjection.
Two, HO-1 introduces the generation of BDF-1 mouse fertilized egg and positive mouse through microinjection
Linearizing target gene fragment is injected in the protokaryon of BDF-1 mouse fertilized egg, makes itself and mouse genome conformity, along with cell constantly divides, each cell will carry this fragment.Newborn mouse was born after 3 weeks, cut off the mouse tail point, carried out the evaluation of transgenic mice with the method for PCR.
Three, transgenosis is at the intravital expression activity of mouse
In conjunction with Fig. 2 A, HO-1 transgenic mice liver HO-1 protein expression level increases.
In conjunction with Fig. 2 B, HO-1 transgenic mice liver HO activity is 28 times of wild-type.
Four, the pathology of transgenic mice, biochemical character
1. the project application people is after successfully having made the HO-1 whole body and having crossed the express transgenic mouse, the tooth of finding above male HO-1 transgenic mice of 10 monthly ages sends red fluorescence (Fig. 3 A, B) when being subjected to uviolizing, and whole male HO-1 transgenic mices all present this phenotype during to 14 monthly ages.
Can send red fluorescence when 2. porphyrin substance is subjected to uviolizing, therefore, point out we above male HO-1 Tg mouse may suffer from certain porphyria at these 10 monthly ages.Through high performance liquid chromatography (high performance liquid chromatography, HPLC) the urine mesoporphyrin content of mouse (be called for short erythrodontia mouse) of red fluorescence is appearred in tooth and the inspection of composition is found, porphyrin content is apparently higher than the brood mouse of wild-type, and is " uroporphyrin (URO)＞hepatacarboxyporphyrin (HEPTA) 〉=coproporphyrin (COPRO) " sample and drains figure.Such porphyrin drainage figure is consistent with PCT patient's.
3. further, pathologic finding confirms all can see in all erythrodontia Mouse Liver the content (Fig. 3 D, E) of pin sample, and this is the peculiar pathological characters of PCT.
To sum up, make a definite diagnosis above male HO-1 transgenic mice of 10 monthly ages and suffer from PCT.The present invention is the PCT model with male HO-1 transgenic mice, can be used for the Study on Pathogenesis to PCT.
In the present embodiment, the implication of abbreviation is as follows respectively:
PCT (porphyria cutanea tarda, porphyria cutanea tarda)
URO-D (uroporphyrinogen decarboxylase, uroporphyrinogen decarboxylase)
HO-1 (Heme Oxygenase-1, heme oxidase-1)
WT (wild type, wild-type)
Tg (transgene, transgenosis)
URO (uroporphyrin, protoporphyrin)
HEPTA (hepatacarboxyporphyrin, 7 carboxyl porphyrins)
COPRO (coproporphyrin, cp)
Specific embodiment described herein only is that the present invention's spirit is illustrated.The technician of the technical field of the invention can make various modifications or replenishes or adopt similar mode to substitute described specific embodiment, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Claims (2)
1. a method of setting up porphyria cutanea tarda (PCT) transgenic mouse model is characterized in that, may further comprise the steps:
Step 1 makes up a linear transgenic sequence, this sequence from 5 ' contain chicken β-actin promotor, cmv enhancer, mouse HO-1 cDNA encoding sequence, termination signal rabbit β-globin poly (A) sequence to 3 ' successively;
Step 2 is used micro-injection method the gene order that makes up is injected C57BL/6/DBA2 (BDF1) mouse fertilized egg;
Step 3 is implanted the zygote in the step 2 in the uterine tube of false pregnancy mouse;
Step 4 produces F0 for transgenic mice, is integrated with exogenous HO-1 expressed sequence in this mouse genome;
Step 5, positive transgenic mice and the hybridization of wild-type BDF1 mouse with obtaining obtain filial generation.
2. a kind of method of setting up porphyria cutanea tarda (PCT) transgenic mouse model according to claim 1, it is characterized in that, the concrete orientation of described structure one linear transgenic sequence is: amplification HO-1 gene order in the mouse genome, be cloned into the pCAGG carrier, HO-1 cDNA is started by chicken β-actin promotor and cmv enhancer, HO-1 connects rabbit β-globin poly (A) termination signal, plasmid is cut through SalI and DraI enzyme, obtains linear transgenic sequence.
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| CN201010253969.1A CN101921795B (en) | 2010-08-16 | 2010-08-16 | Method for establishing porphyria cutanea tarda (PCT) transgenic mouse model |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206054A (en) * | 2020-02-14 | 2020-05-29 | 河北医科大学第三医院 | Construction method of liver HO-1 gene conditional knockout animal model by using CRISPR-Cas9 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295340C (en) * | 2003-08-13 | 2007-01-17 | 上海南方模式生物科技发展有限公司 | Method for extablishing polymorphism adenoid tumour mouse model |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1295340C (en) * | 2003-08-13 | 2007-01-17 | 上海南方模式生物科技发展有限公司 | Method for extablishing polymorphism adenoid tumour mouse model |
Non-Patent Citations (3)
| Title |
|---|
| GAO XU等: "Transgenic overexpression of erythroid-type 5-aminolevulinate synthase causes variegate porphyria in mice: a novel mouse model for acute porphyria", 《ENDOCRINOLOGICAL AND METABOLIC DISEASES》 * |
| J G STRAKA等: "Porphyria and Porphyrin Metabolism", 《ANNUAL REVIEW OF MEDICINE》 * |
| STEFAN W. RYTER等: "THE HEME SYNTHESIS AND DEGRADATION PATHWAYS: ROLE IN OXIDANT SENSITIVITY Heme Oxygenase has Both Pro- and Antioxidant Properties", 《FREE RADICAL BIOLOGY & MEDICINE》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111206054A (en) * | 2020-02-14 | 2020-05-29 | 河北医科大学第三医院 | Construction method of liver HO-1 gene conditional knockout animal model by using CRISPR-Cas9 |
| CN111206054B (en) * | 2020-02-14 | 2022-12-30 | 河北医科大学第三医院 | Construction method of animal model for conditionally knocking out liver HO-1 gene by using CRISPR-Cas9 |
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