CN108938613B - Application of tamoxifen and method for constructing cleft palate animal model - Google Patents
Application of tamoxifen and method for constructing cleft palate animal model Download PDFInfo
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- CN108938613B CN108938613B CN201710390997.XA CN201710390997A CN108938613B CN 108938613 B CN108938613 B CN 108938613B CN 201710390997 A CN201710390997 A CN 201710390997A CN 108938613 B CN108938613 B CN 108938613B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
- A61K31/138—Aryloxyalkylamines, e.g. propranolol, tamoxifen, phenoxybenzamine
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
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Abstract
The invention relates to the technical field of genetic engineering, in particular to application of tamoxifen and a method for constructing a cleft palate animal model. The invention researches and discovers that the Chinese medicine is the female (Tgfbr 1)f/fK14Cre +) and (Tgfbr 1)f/fK14Cre-) mating, or (Tgfbr 1)f/ fK14Cre-) and (Tgfbr 1)f/fK14Cre +) mice, and fetal mice can realize Tgfbr1 gene knockout under the induction of tamoxifen. Thereby realizing the construction of the cleft palate animal model. In the mice constructed by the method provided by the invention, the Tgfbr1 gene in the palatine epithelial cells is knocked out, and the interference of other factors on the cleft palate model can be reduced.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of tamoxifen and a method for constructing a cleft palate animal model.
Background
Since the completion of the human genome project, life has entered scientific research into the post-gene era, and the core work of the post-genome era is to study the functions of genes. Currently, the most effective method for studying gene function is gene targeting (gene knock-out and knock-in) technology. The gene targeting technology is a new technology developed in the field of life science research in recent years, is developed on the basis of an embryonic stem cell technology and a homologous recombination technology, can directionally operate and change genes, and then obtains information of gene functions by observing and analyzing phenotypes of animals with the changed genes. The gene technology has another important application to making animal models of human diseases.
The research shows that many human diseases are caused by gene mutation, and the gene targeting technology can accurately control genes on animal chromosomes so as to produce animal models with the same phenotype as the human diseases, which has very important practical significance for researching the pathogenic mechanism of the human diseases, particularly for searching specific drugs for treating the human diseases. However, the traditional gene knockout can cause deletion mutation of the genome of all cells, often causes serious developmental defect or embryonic death, and is not beneficial to analysis of the gene function at the later development stage, and the current selective gene knockout successfully solves the problem, and can delete a certain gene at a specific time and a specific site, so that the function of the gene and the change of related gene expression can be better understood.
Cleft palate is a congenital craniofacial developmental deformity which is common in newborns, the incidence rate of the newborns is 1/700 in the world, the incidence rate of the newborns in China is 1.82 per thousand, and China is one of the countries with the largest number of cleft lip and palate patients in the world at present. The etiology and mechanism of cleft palate are complex and the pathogenesis and mechanism of cleft palate are unclear. TGFBR1, also known as ALK5(activin receptor like kinase 5), is widely expressed in most cells and specifically responds to TGF-beta signals. The human ALK5 protein consists of 426 amino acids and has a molecular mass of about 55 kD. TGF- β s is a group of polypeptide growth factors involved in embryonic development in many ways, and 3 subtypes have been identified from mammals: TGF-beta 1, TGF-beta 2 and TGF-beta 3, have 70 to 80 percent of homology. The TGF β subtype plays an important role as an endogenous factor in mammalian palatogenesis. The cleft palate model can be obtained by knocking out Tgfbr1 to study the mechanism of cleft palate occurrence. The reported mouse cleft palate model established by Dudas et al and with Tgfbr1 gene knocked out under K14 condition has a soft palate posterior cleft palate phenotype, but the Dudas model has wide range and may have the expression of strong gene function complementation effect to reduce deformity, wherein Tgfbr1 is knocked out in all epithelia.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an application of tamoxifen and a method for constructing an animal model of cleft palate. The method provided by the invention can specifically knock out the Tgfbr1 gene in the epithelial cells of the palate at a specific time, thereby eliminating the interference of other factors on the cleft palate model.
The invention provides application of tamoxifen in preparation of a preparation for knocking out Tgfbr1 gene.
In a specific embodiment of the invention, the Tgfbr1 gene is Tgfbr1 gene in epithelial cells of the palate.
The human keratin 14(K14) promoter as a mammalian villus structural protein is expressed in pairs with keratin 5 in epidermal basal layer cells, villi and hair follicle outer root sheath cells with active nuclear division of transgenic mice, and is a biomarker of stratified squamous epithelium. According to the invention, Cre recombinase is placed under the regulation of a K14 promoter, and tamoxifen is used for starting the expression of Cre recombinase, so that Tgfbr1 gene is knocked out in mouse epithelium.
The invention also provides application of tamoxifen in preparation of a preparation for constructing an animal with a cleft palate phenotype.
In some embodiments, the cleft palate phenotype animal is a mouse. Specifically, the cleft palate phenotype animal is a mouse.
The invention also provides a method for constructing the cleft palate phenotype animal, which comprises the steps of administering tamoxifen to a pregnant animal to obtain the cleft palate phenotype animal;
the genotype of the pregnant animal is Tgfbr1f/fThe female parent and genotype of K14Cre + are Tgfbr1f/fMating K14 Cre-male parents to obtain;
or the genotype is Tgfbr1f/fThe female parent and genotype of K14 Cre-is Tgfbr1f/fAnd the K14Cre + male parents are obtained by mating.
The pregnant animal is a pregnant mouse.
The genotype adopted by the invention is Tgfbrf/fThe K14Cre + mouse is constructed by adopting an embryonic stem cell technology, firstly, a gene sequence which respectively contains a LoxP site at two ends of a Tgfbr1 third exon is constructed in vitro, and then the gene sequence constructed in vitro is transferred into an embryonic stem cell to replace the original gene sequence in a cell genome through homologous recombination. The embryonic stem cells thus treated were re-implanted into the uterus of pseudopregnant miceIn addition, the transgenic mouse is allowed to develop into a complete embryo and finally becomes a transgenic mouse. In this transgenic mouse, LoxP sites were introduced into introns of the corresponding genes, theoretically without affecting the functions of the corresponding genes, and thus the phenotype of the mouse was normal in general.
The genotype adopted by the invention is Tgfbr1f/fK14 Cre-mice are obtained by oocyte injection or embryonic stem cell technology, and in the mice, Cre recombinase is placed under the control of K14 promoter, so that the Cre recombinase can be expressed under a certain condition.
Preferably, the mouse adopted by the invention is subjected to key amino acid mutation in an estrogen ligand binding region, endogenous estrogen can not be bound with the estrogen ligand binding region, and only exogenous estrogen analogue tamoxifen can be bound with the endogenous estrogen ligand binding region, so that the knockout effect of the exogenous estrogen ligand binding region is exerted. The mutation is specifically that the 525 th amino acid of the mouse estrogen receptor is mutated from glycine to arginine.
Specifically, the method for constructing the transgenic mouse used in the present invention is constructed according to the method provided in the literature "Progressive tumor formation with microorganism and with conditional deletion of TGF-beta signaling in head and novel epitope is associated with activation of the PI3K/Akt pathway".
In some embodiments, the pregnant animal is a pregnant mouse, and the administration is started after 9-11 days of pregnancy of the pregnant mouse and is continued for 5 days. Preferably, administration is initiated 10.5 days of pregnancy. The pregnant mice were obtained as follows: at 6 pm, mice were housed 2:1 in their males and females, and the following day, 8 am, were checked for vaginal emboli and weighed, as E0.5 (embryonic day 0.5). And E10.5, weighing again, recording that the person with the weight gain of more than 2g is positive for pregnancy, and starting to give tamoxifen.
In some embodiments, tamoxifen is administered at a concentration of 10mg/mL and at a dose of 100 μ L/day for 5 consecutive days.
In some embodiments, the cleft palate phenotype animal is a fetus of a pregnant animal.
The experiment of the invention shows that under the condition of the concentration of 10mg/mL, tamoxifen is continuously administered for 5 days at the dose of 100 muL/mouse/day, fetal rats show cleft palate, and the results of immunohistochemical staining and RT-PCR research show that the gene expression level of Tgfbr1 is reduced only in palate epithelial cells, and the expression levels of other parts are normal.
The invention also provides a preparation for constructing the cleft palate phenotype animal, which comprises tamoxifen and pharmaceutically acceptable auxiliary materials.
The preparation is in the form of injection, wherein the concentration of tamoxifen is 10 mg/mL.
The invention researches and discovers that the Chinese medicine is the female (Tgfbr 1)f/fK14Cre +) and (Tgfbr 1)f/fK14Cre-) mating, or (Tgfbr 1)f/fK14Cre-) and (Tgfbr 1)f/fK14Cre+)Tgfbr1f/fMice mate, and fetal mice can realize Tgfbr1 gene knockout under the induction of tamoxifen. Thereby realizing the construction of the cleft palate animal model. The invention uses tamoxifen to concentrate the knockout time on the development time of palate as far as possible, and reduces the influence on other tissues. Thus, animals with the Tgfbr1 gene knocked out in the epithelial cells of the palate part are obtained, and the interference of other factors on the cleft palate model is reduced.
Drawings
FIG. 1 shows the results of genotyping of fetal mice by PCR;
FIG. 2 shows the results of phenotypic identification using a stereomicroscope and HE staining; arrows show that the palatal suture of the Cre + fetal mouse is not fused, and the palatal suture of the Cre-fetal mouse is fused;
FIG. 3 shows immunohistochemistry and RT-PCR methods confirmed that TGFBR1 protein is not expressed in palatal epithelial tissue, showing that TGFBR1 protein is deficient there; wherein, FIG. 3-a is an immunohistochemical staining chart of fetal mouse coronary sections; FIG. 3-b shows the expression of TGFBR1 protein in different tissues of fetal rat; FIG. 3-c shows the expression of Tgfbr1mRNA from different tissues of fetal mice.
Detailed Description
The invention provides application of tamoxifen and a method for constructing an animal model of cleft palate, and a person skilled in the art can realize the purpose by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The instruments adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
1. Laboratory animals and materials
1.1 Experimental animals
Experimental rats were given by national institute of dental and jaw cranium (NIDDR/NIH) Dr.Ashock Kulkarni (the construction method is referred to as Progressive formation with mice and conditional deletion of TGF-beta signaling in head and neutral epithelial is associated with activation of the PI3K/Akt path), and the gestation period was about 21 days.
1.2 Experimental drugs
Tamoxifen: purchased from Sigma Co
Sunflower seed oil: purchased from Sigma Co
2. Method of producing a composite material
Genotype at 6 p.m. (Tgfbr 1)f/fK14Cre +) mouse and genotype thereof are (Tgfbr 1)f/ fK14Cre-) mouse cage (or female (Tgfbr)f/fK14Cre-)+♂(Tgfbrf/fK14Cre +)). Mice were male and female 2:1, checked for vaginal emboli the next 8 morning spots and weighed, and scored as E0.5 (embryonic day 0.5). Weighing again at E10.5, recording the person with the weight gain of more than 2g as positive pregnancy, starting injecting Tamoxifen (100 ul/mouse) with the concentration of 10mg/ml, continuously injecting for 5 days, dissecting the pregnant mouse at E16.5, and continuing to close the cage for the negative mouse with the weight less than 2 g.
3. Identification and verification of mouse Tgfbr1 gene conditional knockout cleft palate animal model
3.1 identification of fetal mouse phenotype
3.1.1DNA extraction
(1) 400ul of rat tail lysate was added to 1.5ml EP tube containing rat tail and limbs and digested overnight at 55 ℃.
(2) Add 200ul of saturated 6M NaCl, shake vigorously up and down 200 times, place on ice for 10min, centrifuge at 12000rpm for 10 min.
(3) 500ul of the supernatant was added with 800ul of absolute ethanol, mixed at 12000rpm, and centrifuged for 10 min.
(4) The supernatant was removed and the pellet was washed with 1ml of 75% ethanol, 12000rpm and centrifuged for 10 min.
(5) The supernatant was removed, the precipitate was air dried for 30min, and 50ul ddH2O was added to dissolve the precipitate.
(6) The concentration was measured and recorded.
Rat tail lysate formulation (500 ml): 2.5g of SDS; NaCl: 2.92 g; 7.31g of EDTA; Tris-HCl: 0.6057 mg; proteinase K (PK) 50mg
3.1.2DNA amplification
(1) Primer design
a.Cre 700bp
5'to 3':GCC TGC ATT ACC GGT CGA TGC AAC GA (Cre-A)
5'to 3':GTG GCA GAT GGC GCG GCA ACA CCA TT (Cre-B)b.Tgfbr1 250bp
5'to 3':ACC CTC TCA CTC TTC CTG AGT (ONE-R1)5'to 3':ATG AGT TAT TAG AAG TTG TTT (TWO-R1)
GGA ACT GGG AAA GGA GAT AAC (THEE-R1) (2) Polymerase Chain Reaction (PCR) PCR
Cre reaction System 20ul
Reaction procedure:
Reaction procedure:
3.1.3 electrophoresis
(1) 1% gel preparation 50ml
a. 50ml of agarose gel was weighed out with 0.5g of agarose powder and 50 XTAE diluted to
b. Melting gel the weighed agar powder was mixed with 1 × TAE in a conical flask and heated in a microwave oven 2-3 times until the agarose was completely dissolved.
c. After the sol was cooled to about 50 ℃, 5ul of gold-based view nucleic acid dye was added.
d. And placing a comb in the glue pouring groove, pouring the gel cooled to be not scalding into the rubber plate, standing at room temperature for 30min, slightly pulling out the comb after complete solidification, and forming sample holes which are mutually separated on the rubber plate.
e. Putting the prepared colloid into an electrophoresis tank of 1 XTAE working solution for use, and enabling the prepared colloid to be more than 1mm above the surface of the colloid.
(2) Sample loading in the sample hole: 5ul of DNAmarker was added to the first well, followed by 8ul of sample.
(3) Glue running: and opening the electrophoresis apparatus, determining that the installation of the anode and the cathode is correct so as to prevent the sample from running out of the rubber plate, and carrying out electrophoresis at constant voltage of 120V.
(4) And (3) analysis: and (3) putting the gel plate after electrophoresis into an ultraviolet analyzer, and taking a picture for analysis by a Jetta gel imaging system.
3.2 Total RNA extraction and qRT-PCR of palatine Process tissue
3.2.1RNA extraction
a. Adding liquid nitrogen into a mortar, grinding the tissue into powder in the liquid nitrogen, taking 50-100 mg of tissue powder by using a medicine spoon precooled by the liquid nitrogen, adding the tissue powder into an EP (ultra Violet) tube already containing 1ml of Trizol liquid (note that the total volume of the tissue powder cannot exceed 10% of the volume of the used Trizol), and fully and uniformly mixing.
b. After 5min at room temperature, 200ul of chloroform was added, the EP tube was closed and shaken vigorously for 15 seconds.
c.12000rpm centrifugation for 10min, taking the upper aqueous phase in a new EP tube (ten million don't mix the middle precipitation layer and the lower liquid, otherwise re-centrifugation), adding 500ul isopropanol, and mixing by gentle inversion. Standing at room temperature for 10min, and centrifuging at 12000rpm for 10 min.
d. The supernatant was carefully discarded, 1ml of 75% ethanol was added, vortexed and mixed, and centrifuged at 12000rpm for 5min at 4 ℃. The operation was repeated once.
e. The supernatant is discarded (residual liquid is removed as much as possible), and dried for 5-10 min at room temperature or under vacuum (care is taken not to dry too much, otherwise the solubility of RNA is reduced). Dissolving RNA with 30ul DEPC treated water, and if necessary, carrying out water bath at 55-60 deg.C for 10min, and refrigerating at-80 deg.C for use.
(2) Primer design
a.Tgfbr1 215bp
CAGCTCCTCATCGTGTTGGTG from upstream 5' to 3
Downstream 5'to 3' GCACATACAAATGGCCTGTCTC
b.β-actin 233bp
GTTGGTTGGAGCAAACATCC from upstream 5' to 3
Downstream 5'to 3' AAGCAATGCTGTCACCTTCC
(3) Reverse transcription PCR (20ul System)
a. The following components are added into a microcentrifuge tube according to requirements, and the total amount is 12ul
10 μ L of total RNA;
random primer (500ug/ml) 100 ng;
UdNTP(10mM) 1μL；
ddH2O made up to 12. mu.L.
b. Heating the above mixture at 65 deg.C for 5min, rapidly cooling on ice, centrifuging for a short time, and adding the following components:
5 Xfirst Strand Synthesis buffer 4. mu.L
0.1M DTT 2μL
RNaseInhibitor 1μL。
c. The components in the centrifuge tube were gently mixed and incubated at 37 ℃ for 2 min.
d. Add 1ul (200U) M-MLV reverse transcriptase, gently blow and mix well, if random primers are used, incubate centrifuge tubes for 10min at 25 ℃.
e.37 ℃ incubation for 50min, 70 ℃ heating for 15min to terminate the reaction.
(4)Real-time PCR
Configuring a Real-time PCR reaction system:
real-time PCR reaction program
3.2 tissue section preparation
Decalcified tissue is washed overnight with running water
(1) And (3) dehydrating: gradient alcohol (70%, 80%, 90%, 100%, 100% each 2 hours)
(2) And (3) transparency: xylene (twice in 30 min)
(3) Embedding: the treated tissue was placed in an embedding machine opened in advance (paraffin melted at 60 ℃ C.), and 1 hour of paraffin immersion was carried out, and the coronal section was vertically embedded.
(4) Slicing: fixing the specimen in a microtome, continuously slicing at coronal position, baking at 60 deg.C for 5 hr to a thickness of 4 μm, cooling to room temperature, and loading into a cassette.
3.3HE staining
(1) Slicing, baking at 65 deg.C for 1 hr
(2) Xylene, 5 min. times.3
(3) 100%, 95% ethanol, 2 times each for 5min
(4) Washing with distilled water for 2 times
(5) Hematoxylin 8 min, tap water washing 2 times
(6) Alcohol differentiation with hydrochloric acid for 10s, adding bluing liquid (under-mirror observation control)
(7) Eosin for 2min, distilled water and 95% ethanol.
(8) 100% ethanol, 3min/2 times
(9) Xylene transparent, 5min/3 times
(10) Sealing neutral gum, air drying, and observing under the mirror
3.4 immunohistochemical staining
(1) The paraffin section dewaxing rehydration is the same as the paraffin section dewaxing rehydration before HE dyeing.
(2)3％H2O2And (5) incubating for 5-10 minutes at room temperature to eliminate the activity of endogenous peroxidase. PBS was washed 3 times for 5 minutes each.
(3) Blocking with 10% normal goat serum (PBS dilution) and incubating at room temperature for 1 hour. The serum was decanted, washed free, and diluted primary antibody was added dropwise in appropriate proportions and the freezer was kept overnight at 4 ℃.
(4) PBS washing, 5 minutes x 3 times, dripping the horseradish enzyme labeled secondary antibody (PBS dilution) diluted by proper proportion, and incubating for 30 minutes at 37 ℃.
(5) PBS washing, 5 minutes multiplied by 3 times, DAB kit color development, tap water washing, hematoxylin counterstaining for 10s, absolute ethyl alcohol dehydration, xylene transparency, neutral gum sealing, airing and observation under a mirror.
4, experimental results:
1. cleft palate fetal rat genotype identification
The detection is carried out by using K14-Cre primer, the size of Cre gene fragment is 700bp, and the existing Cre of filial generation mice can be seen from figure 1+The gene also has Cre-Phenotype of the gene, but only Cre+Knockout of the Tgfbr1 gene was only possible in fetal mice after tamoxifen injection. The results show that Cre is obtained+The probability of fetal rats is about 50%.
2. Stereomicroscope and HE staining results
Under a stereoscopic microscope, 60% of Cre is given to tamoxifen (concentration 100mg/mL, each 100. mu.L each day, for 5 consecutive days in pregnant mice)+The distance between the palatoschis on both sides of the fetal rat is far, and the oral cavity and the nasal cavity are directly communicated, so that the fetal rat is expressed as a cleft palate phenotype. And Cre-Most of the palatine compartment of the fetal rat had fused, and a small cleft was seen in 1/3 of the posterior palatine compartment. HE staining visible Cre+The fetal rat has enlarged tongue body, occupies most oral cavity, and palatine process has rotated to the upper part of tongue body and grows to horizontal direction, but two lateral palatine processes do not contact, i.e. cleft palate occurs, Cre-The tongue of the fetal rat is changed into a flat shape,descending into oral cavity, lifting palatine process to the upper part of tongue body, growing horizontally and fusing, sealing oral cavity, and fusing palatine parts to form obvious incompletely degenerated mesolamella without cleft palate (fig. 2). In addition, experiments have shown that tamoxifen (concentration 100mg/mL, 50 or 75 μ L each per day, continuous administration to pregnant mice for 5 days) in Cre is administered+Fetal mice do not exhibit the cleft palate phenotype.
3. Immunohistological staining and RT-PCR results
To detect Tgfbr1 gene knockout, we identified by immunohistochemical staining and qRT-PCR results that Cre with cleft palate phenotype was found as a result of immunohistochemical analysis+The immunohistochemical staining of the fetal mouse coronary section TGFBR1 protein is obviously better than that of Cre-The fetal mice are shallow, and Cre is found after the analysis of the fetal mice by Image Pro Plus analysis software+The expression of the fetal rat palatine epithelial tissue TGFBR1 protein is obviously lower than that of Cre-Fetal rat (P)<0.01) and tissues in other parts of the craniofacial area showed no difference in TGFBR1 protein expression in immunohistochemical staining. The qRT-PCR result shows Cre with cleft palate phenotype+The gene expression quantity of the fetal rat palatine Tgfbr1 is obviously lower than that of Cre-Palatine process of fetal rat (P)<0.01), which fully indicates that the Cre/Loxp system can effectively knock out the Tgfbr1 gene of the palate. (FIG. 3).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.
Claims (2)
1. A method of constructing an animal with a cleft palate phenotype, comprising administering to a pregnant animal tamoxifen to obtain an animal with a cleft palate phenotype;
the genotype of the pregnant animal is Tgfbr1f/fThe female parent and genotype of K14Cre + are Tgfbr1f/fMating K14 Cre-male parents to obtain;
or the genotype is Tgfbr1f/fThe female parent and genotype of K14 Cre-is Tgfbr1f/fMating K14Cre + male parents to obtain; the concentration of tamoxifen administrationThe dose is 10mg/mL and the dose is 100 mu L/day; the pregnant animal is a pregnant mouse, administration is started after 9-11 days of pregnancy of the pregnant mouse, and administration is continuously carried out for 5 days.
2. The method of claim 1, wherein said cleft palate phenotype animal is a fetus of a pregnant animal.
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| WO2008066634A2 (en) * | 2006-11-01 | 2008-06-05 | Jennings-Spring Barbara L | Method of treqatment and compositions of d-chriro inositol and phosphates thereof |
| CN103494776A (en) * | 2013-07-22 | 2014-01-08 | 南通广泰生化制品有限公司 | Tamoxifen citrate freeze-dried powder injection |
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| CN103494776A (en) * | 2013-07-22 | 2014-01-08 | 南通广泰生化制品有限公司 | Tamoxifen citrate freeze-dried powder injection |
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