CN104593418A

CN104593418A - Method for establishing humanized rat drug evaluation animal model

Info

Publication number: CN104593418A
Application number: CN201510061140.4A
Authority: CN
Inventors: 张连峰; 马婧
Original assignee: Institute of Laboratory Animal Science of CAMS
Current assignee: Institute of Laboratory Animal Science of CAMS
Priority date: 2015-02-06
Filing date: 2015-02-06
Publication date: 2015-05-06

Abstract

The invention provides a method for establishing a humanized rat drug evaluation animal model. According to the method, a multidrug resistance gene 1 (Abcb1)-knocked-out genetically engineered rat is obtained through a microinjection method by virtue of a CRISPR/Cas9 gene knockout technology and 153kb bacterial artificial chromosome (BAC) fragments containing a humanized Abcb1 promoter and cDNA is simultaneously inoculated into the rat genome through the microinjection method by virtue of a large fragment transgenic technology to obtain a transgenic rat capable of stably expressing human Abcb1 and the genetically engineered rat and the transgenic rat are hybridized to establish the humanized rat drug evaluation animal model. RT-PCR analysis shows that Abcb1 expression profiles of humanized Abcb1 rat are significantly different from those of the rat endogenous Abcb1. The method has the beneficial effects that the humanized rat capable of expressing human Abcb1 is obtained and the rat is used for expressing human Abcb1 genes and has closer expression profiles to those of human so that the model can be well used for the efficacy evaluation of newly developed drugs.

Description

A kind of method of humanization rat drug evaluation Animal Model

Technical field

The present invention relates to biological technical field, be specifically related to a kind of method of humanization rat drug evaluation Animal Model.

Background technology

Can produce an original new drug in average 1 ten thousand to 25 thousand isolated compound, cost is up to 10-17 hundred million dollars.Cause large quantities of lead compound to be eliminated and in reason that drug candidate research and development stop except pharmacodynamics significantly except, medicament research and development close to 50% stops owing to dealed with medicine went, because the toxicity of drugs on patients also once went out not poor from the event of market withdrawal medicine, not only cause huge financial loss, also cause the loss of life.Therefore, international pharmaceutical industry studying always new drug development commitment to new chemicals carry out toxicity screening accurately and reliably, the method system of rapid sensitive, thus Timeliness coverage and superseded chemicals or the chemical structure having genotoxic potential, shorten the time of new drug development as far as possible and reduce cost and risk.

Present toxicity detection system, comprises the Monitoring techniques to drug toxicity, cell model and the animal model toxicity prediction to medicine unsatisfactory.This is also one of predicament of facing of current international pharmaceutical industry toxicity prediction.In animal model, mainly because animal is in the gene of process and the species variations of the mankind such as drug absorption, transhipment, metabolism, removings, comprise gene structure, express spectra, regulation and control, and the difference to drug toxicity reaction do not caused on an equal basis of number gene.Such as, troglitazone (Troglitazone) is the oral medicine for the treatment of diabetes, and results of animal is very good.But FDA receives many cases liver toxicity clinical report very soon after coming into the market.This medicine is withdrawn from market by FDA in March, 2000, at present only in the field of study as test positive drug.Between 2005-2010 6 years, just there are more than 10 because the toxicity such as liver, kidney, nerve, heart in patient's use is withdrawn typical medicaments, without causing huge financial loss, also lived loss.

So, with the corresponding gene of the gene substitution animal of the process such as drug absorption, transhipment, metabolism, removing of the mankind, setting up the model that gene structure, express spectra and regulative mode are similar to the mankind, i.e. humanized animal's model, is one of effective way addressed this problem.The time shortening new drug development is reduced cost and risk and had great importance.

The gene that medicine is relevant can be divided into drug metabolism involved enzyme, and the membrane channel that medicine combines relevant acceptor, drug transport is correlated with, the genes involveds such as intracellular signaling more than 100 are planted.More external pharmaceutical companies have started the research of human medicine genes involved humanization mouse, and have developed CYP3ACluster, CAR, AHR etc. more than ten plant drug gene humanization mouse, and are applied to the research of drug metabolism and safety evaluation.At present the patent of these humanization mouse is all abroad in some pharmaceutical companies hands, limits the use of China's medical research organisations.In addition, mouse is not the drug evaluation animal of CFDA accreditation, in the difference and gene regulating of express spectra, still have larger difference with the mankind.From the scientific meaning of drug evaluation, build the humanized model that the CFDA such as rat specify, have more wide demand.And along with the maturation of TALEN and CRISPR/Cas9 genome editing technique, genomic modification no longer relies on the gene targeting of embryonic stem cell, makes the humanization of rat become possibility.The states such as rat becomes " favorite " of biological medicine research again, American-European are operating extensive rat model the Study on Resources, and the intellecture property of rat model and the Medicine innovation achievement controlling to drive thus are seized in attempt.

P-glycoprotein to be a kind of relative molecular weight be 170 000 single transmembrane glycoprotein, encoded by multidrug resistance gene Abcb1 (also known as Mdr1), be found in the earliest in tumour cell, but research subsequently shows that it is extensively present in each tissue of body, participate in the processes such as drug absorption, distribution, metabolism and excretion.P-glycoprotein can utilize the ATP in body to transport out by the ectogenic medicine poisonous substance entering cell, reduces Intracellular drug concentration of poisons.Under physiological status; the existence of P-glycoprotein can protect organism safe; and in pathological conditions; P-glycoprotein makes medicine not enter central nervous system in the expression of hemato encephalic barrier; expression in tumour cell causes body and produces multi-drug resistance phenomenon; thus have impact on the treatment of disease, so the Abcb1 gene of control P-P-glycoprotein expression is relevant with security to multi-medicament validity.Therefore dosage choice and evaluating drug effect that Abcb1 gene humanization rat model can perform well in medicine newly developed is built.

Summary of the invention

The object of this invention is to provide a kind of construction process of Abcb1 gene humanization rat model.

Key of the present invention is that utilizing CRISPR/Cas9 gene Knockout to prepare rat gene respectively knocks out, utilizes the large segment transgenic technology of BAC to build transgenic rat model.

The preparation method of this humanization rat comprises the following steps:

One: the structure being cloned into bacterial artificial chromosome BAC containing the most long pruning junctor cDNA of mankind Abcb1 gene and the regulating and controlling sequence of upstream and downstream thereof;

Two: microinjection rat male pronucleus, obtain positive in mankind Abcb1 transgenic rat, it comprises the most long pruning junctor cDNA of mankind Abcb1 gene and the regulating and controlling sequence of upstream and downstream thereof, called after hAbcb1-BAC positive rats;

Three: for making the sgRNA plasmid construction of Abcb1 knockout rat;

Four: by sgRNA and Cas9 mRNA hybrid injection SD rat male pronucleus, obtain positive Abcb1 gene and knock out rat, called after rAbcb1 knockout rat;

Five: rAbcb1 knockout rat male and female are handed over mutually and obtains rAbcb1 gene knockout homozygote rat;

Six: the intermolecular hybrid of hAbcb1-BAC positive rats and rAbcb1 knockout rat can be obtained the BAC positive and rAbcb1 gene knockout heterozygote rat;

Seven: by BAC, positive and rAbcb1 gene knockout heterozygote rat and rAbcb1 gene knockout homozygote rat hybridize, and can obtain the hAbcb1-BAC positive and rAbcb1 gene knockout homozygote rat.The inherent gene level of this rat body has lacked the expression of self Abcb1 gene, meanwhile, it also carries the Abcb1 gene of the mankind obtained by BAC technology, i.e. Abcb1 humanization rat.

Accompanying drawing explanation

Fig. 1 is the structure iron of BAC-Abcb1 bacterial artificial chromosome.

Fig. 2 is that the transgenic rat PCR of the Abcb1 expressing people identifies electrophorogram.M:Marker DL2000 (Takara); H:DNA template is water; W:DNA template is wild crt gene group DNA; 1-15 is that F0 numbers for rat.

Fig. 3 is that Abcb1 knockout rat PCR identifies electrophorogram.M:Marker DL2000 (Takara); H:DNA template is water; W:DNA template is wild control group genome; 1-12 is that the F0 produced by microinjection is numbered for rat.

Fig. 4 is that the PCR of Abcb1 gene humanization rat identifies electrophorogram.P:DNA template is positive reference substance.1-6: knock out rat by BAC-Abcb1 rat and Abcb1 b and hybridize the filial generation produced.A:2,4,6 is transgenic positive mouse; 1,3, No. 5 is transgene negative mouse.B.1, No. 2 is wild-type rats; 3, No. 4 is Abcb1b gene knockout heterozygote rat; 5, No. 6 is Abcb1b gene knockout homozygote rat.No. 6 is Abcb1 humanization rat.

Fig. 5 is mouse source and people source Abcb1 m rna expression level in wild-type rats and humanization rat different tissues.

Embodiment

Below in conjunction with specific embodiment, the present invention is further illustrated.Should be understood that these embodiments only for illustration of the present invention but not for limiting scope of the present invention.

Embodiment 1: the making expressing the transgenic rat of the bacterial artificial chromosome of Abcb1

By the regulating and controlling sequence of the most long pruning junctor cDNA (4718bp) of mankind Abcb1 gene and upstream and downstream thereof, (upstream length is 66kbp, ATG downstream length is 87kbp), be cloned in bacterial artificial chromosome BAC, complete the BAC expression vector of expressing Abcb1.By the carrier that builds after the extracting of phenol chloroform method, adjustment concentration, to 1-2ng/ μ l, utilizes microinjection technique to be expelled to by BAC in the zygote of SD rat, with SD rat as pseudopregnant recipients rat, prepares transgenic rat.Obtain 15 F0 altogether for rat, extract the genomic dna of birth rat, for detecting its integrity, three pairs of PCR primer are used to carry out integrity detection to the upper, middle and lower reaches of inserting BAC respectively, be respectively: Abcb 1-BAC-R 1:5'-GCTGCTGGTGCTGATTGT-3'Abcb1-BAC-F2:5'-CCAAATGCTGCTCA AG-3' downstream, Abcb1-BAC-F1:5'-CACCAGTAAGAGCGTTGA-3' downstream Abcb 1-BAC-R2:5'-GAGTTTATGCCACCAAGTAG-3'Abcb1-BAC-F3:5'-AGTGG TTTCAGAATGGC-3' downstream Abcb 1-BAC-R3:5'-GTCAGTTACAGTAAATGGG-3'.PCR reaction system 20 μ l.Reaction conditions: 95 DEG C of 5min; (95 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations); 72 DEG C of 10mins; 4 DEG C of insulations.Amplified fragments size is respectively 316bp, 449bp, 410bp.Be tested and appraised, obtain 4 altogether and be merely able to express the transgenic rat of total length by subsequently these 4 head being built mouse and wild-type mating, analyze its situation that goes down to posterity, discovery F10 can not go down to posterity, and 2,5,8 complete fragment and stablizing go down to posterity.

The making of embodiment 2:Abcb1 knockout rat

GRNA plasmid construction for Abcb1 gene targeting: a target spot GGAGACAAATACACAAGATT for Abcb1 gene design, synthesizes a pair oligonucleotide chain (TAGGAGACAAATACACAAGATT and AAACAATCTTGTGTATTTGTCT; For the preparation of the oligonucleotide of sgRNA. synthesis through annealing (naturally being down to room temperature after 97 DEG C of 6mins), be connected into the pUC57-sg rna expression carrier cut back to close through Bsa I enzyme, structure sgRNA expression vector.Whether be correctly connected into fragment by sequence verification, select correct clone, prepare after enlarged culturing to be used for later stage in-vitro transcription.

In-vitro transcription: Cas9 expression plasmid, through Age I linearization for enzyme restriction, after phenol chloroform purifying, is dissolved in as template in the water of nuclease free, for in-vitro transcription.The synthesis of Cas9 mRNA acts on T7 RNA polymerase in vitro by test kit T7Ultra Kit (Amibion, AM1345) and completes.The expression vector of sgRNA, after DraI linearization for enzyme restriction, through phenol chloroform purifying, is dissolved in as template in the water of nuclease free, for in-vitro transcription.The external synthesis of sgRNA utilizes T7 RNA polymerase to complete by test kit MEGAshortscript Kit (Ambion, AMI354) in vitro.

The procaryotic injection of Cas9/sgRNA: Cas9 mRNA and sgRNA transcribed mixes and adjust concentration to 10 ng/ μ l and 5ng/ μ l/sgRNA, in the male nucleus that RNA mixture is expelled to the zygote of SD rat by microinjection and tenuigenin, with SD rat as pseudopregnant recipients rat, 12 rats are obtained by microinjection, pass through genotype identification, as can be seen from the figure 1,5, there is notable difference in 9 three rats compared with wild type band.We analyze 12 rat target spot amplified productions subsequently, finally determine by TA clone, order-checking the genetic modification situation that CRISPR/Cas9 causes Abcb1.

Embodiment 3: the cultivation of humanization Abcb1 rat

By the intermolecular hybrid of the rAbcb1 obtained knockout rat can be obtained rAbcb1 gene knockout homozygote rat, by the intermolecular hybrid of hAbcb1-BAC positive rats and rAbcb1 knockout rat being obtained the BAC positive and rAbcb1 gene knockout heterozygote rat, again by this strain rats and rAbcb1 gene knockout homozygote rat being hybridized, the hAbcb1-BAC positive can be obtained and rAbcb1 gene knockout homozygote rat.The inherent gene level of this rat body has lacked the expression of self Abcb1 gene, meanwhile, it also carries the Abcbl gene of the mankind obtained by BAC technology.

The expression analysis of embodiment 4:Abcb1 humanization rat

The rat of the genotype identification positive, by with wild-type mating, go down to posterity situation and expression of the gene of its filial generation is analyzed.Cervical dislocation sacrifices rat, extracts the total serum IgE of the heart of three F1 generation rats (being built the positive mouse of mouse and wild type crosses gained by each head) and wild control rats rat, liver, kidney, lung, cerebral tissue.The total serum IgE extracted utilizes Reverse Transcriptase kit (Life Technologies) reverse transcription to become cDNA, primer RT-Abcb1-F:5'-GGCTATCATTACTCTTTACCTGTGAAG-3' and the RT-Abcb1-R:5 '-CCGGATTGACTGAATGCTG-3' size that increases is utilized to be the object fragment of 225bp, PCR reaction system 20 μ l.Reaction conditions: 95 DEG C of 5min; (95 DEG C of 30s, 62 DEG C of 30s, 72 DEG C of 30s, 24 circulations); 72 DEG C of 10mins; 4 DEG C of insulations.GAPDH, as internal reference, determines the expression of Abcb1 in five kinds of tissues.

Claims

1. for the preparation of the bacterial artificial chromosome BAC expression vector needed for stably express people Abcb1 transgenic rat.

2. expression vector constructs according to claim 1, wherein genome sequence comprises the most long pruning junctor cDNA of mankind Abcb1 gene.

3. expression vector constructs according to claim 1, wherein genome sequence comprises mankind Abcb1 upstream region of gene control region, the control region in promoter region and downstream.

4., for the preparation of the sgRNA expression vector needed for the knockout rat knocking out Abcb1, the plasmid that it is characterized in that setting out is PUC57.

5. vector construct according to claim 4, is characterized in that wherein comprising following two sgRNA sequence: TAGGAGACAAATACACAAGATT and AAACAATCTTGTGTATTTGTCT.