Ces2 gene knockout rat model and construction method and application thereof
阅读说明：本技术 Ces2基因敲除大鼠模型及其构建方法和应用 (Ces2 gene knockout rat model and construction method and application thereof ) 是由 王昕� 尚旭阳 鲁健 张远金 于 2020-06-12 设计创作，主要内容包括：本发明公开了一种用于药物代谢研究的Ces2敲除大鼠模型的构建方法及其应用。所述Ces2敲除大鼠模型的构建方法包括：基因敲除靶点的选择、sgRNA合成、胚胎显微注射、大鼠饲养与繁殖等步骤,得到纯合子Ces2基因敲除大鼠模型。然后对纯合子Ces2基因敲除大鼠模型从mRNA水平进行Ces2表达检测,以及代谢功能验证,以证明该Ces2基因敲除大鼠模型构建成功。本发明利用上述方法成功构建Ces2a/j、Ces2c及Ces2a/c/j基因敲除大鼠模型,该模型不仅可以作为Ces2相关的药物代谢研究的重要工具,还是研究Ces2生理功能的重要动物模型。(The invention discloses a construction method and application of a Ces2 knockout rat model for drug metabolism research. The construction method of the Ces2 knockout rat model comprises the following steps: selecting a gene knockout target, synthesizing sgRNA, performing embryo microinjection, feeding and breeding rats and the like to obtain a homozygote Ces2 gene knockout rat model. Then, Ces2 expression detection and metabolic function verification are carried out on a homozygote Ces2 gene knockout rat model from the mRNA level, so that the Ces2 gene knockout rat model is successfully constructed. The invention successfully constructs the Ces2a/j, Ces2c and Ces2a/c/j gene knockout rat model by using the method, and the model not only can be used as an important tool for researching drug metabolism related to Ces2, but also can be used as an important animal model for researching the physiological function of Ces 2.)
1. A method for constructing a rats model of Ces2 gene knockout, which comprises the following steps:
(1) determining a knockout target point;
the knockout targets are two 18bp DNA sequences taking NGG as the tail;
(2) in vitro synthesis and transcription of a sgRNA double-stranded template;
the sgRNA double-stranded template is obtained by amplifying an oligonucleotide sequence Oligo with the length of 60bp, which comprises a T7 promoter sequence and an 18bp target sequence, through a PCR reaction;
(3) embryo microinjection and transplantation;
co-injecting the sgRNA and Cas9 mRNA into fertilized egg cytoplasm, and transplanting the fertilized egg into an oviduct of a pseudopregnant female mouse;
(4) constructing a Ces2 gene knockout rat model;
breeding a pseudopregnant mother rat to obtain an F0 generation single-gene or multi-gene heterozygote rat model, breeding an F0 generation single-gene or multi-gene heterozygote rat model and a wild type rat in a cage to obtain a progeny which is an F1 generation single-gene or multi-gene heterozygote rat model, breeding an F1 generation rat with the same genotype in a cage to obtain a homozygote mutant rat, namely an F2 generation Ces2 gene knockout rat model.
2. The method of claim 1, wherein in step (1), the target of knockout is two 18bp DNA sequences ending in NGG that are 50bp apart; and/or in the step (2), the sgRNA injection concentration is 20-40 ng/mu L; and/or, in step (2), the Cas9 mRNA injection concentration is 30-60 ng/. mu.L.
3. The method of claim 1,
in the step (1), the target points are as follows:
ces2 a: TTGGCTAGACTTCCTGGT (SEQ ID NO.1) and TCTCCTCCAGCATGTGCA (SEQ ID NO. 2);
ces2 c: CGGAAACAACCACATAGC (SEQ ID NO.3) and TATTCTTATCCATGTGTG (SEQ ID NO. 4);
in the step (2), the oligonucleotide sequence Oligo comprises:
Ces2a sgRNA
Oligo1:
GATCACTAATACGACTCACTATAGGGCCTTTGGCTAGACTTCCGTTTTAGAGCTAGAAAT(SEQ IDNO.5)；
Oligo2:
GATCACTAATACGACTCACTATAGGTCTCCTCCAGCATGTGCAGTTTTAGAGCTAGAAAT(SEQ IDNO.6)；
Ces2c sgRNA
Oligo1:
GATCACTAATACGACTCACTATAGGCGGAAACAACCACATAGCGTTTTAGAGCTAGAAAT(SEQ IDNO.7)；
Oligo2:
GATCACTAATACGACTCACTATAGGTATTCTTATCCATGTGTGGTTTTAGAGCTAGAAAT(SEQ IDNO.8)。
4. the method of claim 1, wherein the Ces2 gene knockout comprises a knockout of one or more Ces2 genes.
5. The method of claim 1, wherein in step (2), the sequence of the sgRNA double-stranded template is as follows:
Ces2a sgRNA
template 1:
GATCACTAATACGACTCACTATAGGTTGGCTAGACTTCCTGGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.9)；
template 2:
GATCACTAATACGACTCACTATAGGTCTCCTCCAGCATGTGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.10)；
Ces2c sgRNA
template 1:
GATCACTAATACGACTCACTATAGGCGGAAACAACCACATAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.11)；
template 2:
GATCACTAATACGACTCACTATAGGTATTCTTATCCATGTGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.12)。
6. the method of claim 1, wherein step (4) further comprises the step of genotyping a Ces2 knockout rat model: extracting genome DNA of a rat model, designing primer pairs according to upstream and downstream sequences of a target spot by taking the genome DNA as a template to perform PCR reaction, performing sequencing analysis on PCR products, and selecting rats subjected to non-triplex multiple gene editing at a single site to perform subsequent breeding according to the sequencing analysis result.
7. The method of claim 6, wherein the primer pairs are as follows:
Ces2a
upstream: CTGCTGGCTATTGGCTTCC (SEQ ID NO. 13);
downstream: CCTGCTGCTTTCCATCCC (SEQ ID NO. 14);
Ces2c
upstream: AACTTTCACCCACGACAT (SEQ ID NO. 15);
downstream: AAGGTTACCATCAGTGCC (SEQ ID NO. 16).
8. A preparation method of a Ces2 gene-deleted rat liver microsome is characterized by comprising the following steps: CO 22After the Ces2 knockout rat model prepared by the method of any one of claims 1 to 7 was sacrificed by asphyxiation, the liver of the Ces2 knockout rat model was homogenized and centrifuged at a different speed to obtain the liver microsome of the Ces2 gene-deficient rat.
9. The Ces2 gene-deleted rat liver microsomes prepared by the method of claim 8.
10. Use of a Ces2 knockout rat model prepared by the method of any one of claims 1-7 or of a Ces2 gene-deleted rat liver microsome of claim 9 for in vitro drug metabolism, drug toxicity assessment and compound screening.
11. Use of a rats knockout Ces2 gene model prepared according to any one of claims 1-7 for the study of lipid metabolism in the liver.
12. A method for breeding a multi-subtype Ces2 gene knockout rat model, characterized in that two Ces2 single-gene or multi-gene homozygote knockout rat models prepared by any one of claims 1 to 7 are subjected to cage mating to obtain a double-heterozygote or multi-heterozygote progeny rat, and then the double-heterozygote or multi-heterozygote progeny rat with different genders in the same nest is subjected to selfing to obtain a progeny homozygote knockout rat, wherein the progeny homozygote knockout rat comprises a Ces2 gene knockout rat model.
A sgRNA double-stranded template, wherein the sequence of the sgRNA double-stranded template is as follows:
Ces2a sgRNA
template 1:
GATCACTAATACGACTCACTATAGGTTGGCTAGACTTCCTGGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.9)；
template 2:
GATCACTAATACGACTCACTATAGGTCTCCTCCAGCATGTGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.10)；
Ces2c sgRNA
template 1:
GATCACTAATACGACTCACTATAGGCGGAAACAACCACATAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.11)；
template 2:
GATCACTAATACGACTCACTATAGGTATTCTTATCCATGTGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.12)。
14. use of the sgRNA of claim 13 in constructing a Ces2 knockout rat model.
15. A primer pair, characterized in that the primer pair is as follows:
Ces2a
upstream: CTGCTGGCTATTGGCTTCC (SEQ ID NO. 13);
downstream: CCTGCTGCTTTCCATCCC (SEQ ID NO. 14);
Ces2c
upstream: AACTTTCACCCACGACAT (SEQ ID NO. 15);
downstream: AAGGTTACCATCAGTGCC (SEQ ID NO. 16).
16. Use of the primer pair of claim 15 for constructing a rats model of Ces2 knock-out gene.
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a construction method and application of a carboxylesterase 2(Ces2) gene knockout rat.
Background
Carboxylesterase (CES) is an α/β serine-folded protein, containing multiple genes, belonging to the class B esterases. Plays an important role in the biotransformation of various endogenous and exogenous substances and the activation of prodrugs, is mainly distributed in liver and intestinal tract, has a small amount of distribution in organs such as kidney, lung, brain and the like, and is an important I-phase drug metabolic enzyme in human bodies. CES1 and CES2, as the major subtypes of carboxylesterases, share 47% amino acid sequence similarity, but have a clear propensity for substrate selection. Most of the CES1 substrates have larger acyl groups and smaller alcohol groups, whereas CES2 prefers to select compounds with smaller acyl groups and larger alcohol group structures as substrates. CES2, the most prominent carboxylesterase subtype in the gastrointestinal tract, mediates the first perhydrolysis metabolism of most oral prodrugs and participates in the metabolism of various clinically common antiplatelet drugs, angiotensin receptor antagonists, antiviral drugs and tumor treatment drugs.
Drug metabolism with CES2 involves two modes: metabolic activation and metabolic elimination. Many of the clinically common prodrugs are metabolically activated via CES2, such as the oncology medications irinotecan, LY2334737 (a prodrug of gemcitabine), capecitabine, the angiotensin receptor antagonists candesartan, olmesartan, and the like. Irinotecan is metabolized by CES2 to produce the active metabolite SN-38, which is 1000 times more cytotoxic than irinotecan. Meanwhile, CES2 is also involved in the metabolic clearance of many clinically used drugs, such as flutamide, which is an anti-tumor drug. In addition, many of the drugs that affect the nerve center, such as ***e and heroin, are also metabolized via CES 2. For many reasons, such as ethics, many experiments are performed with the help of experimental animals. Through different research purposes and research methods, one will select appropriate experimental animals to perform the experiment. Rodents are often the first choice animal models in medical research because of their small size, ease of operation, faster reproduction, and higher similarity of genes to humans.
The CES2 family in human has only one subtype, is located on chromosome 16, has a full length of 10.9kb, the Ces2 subtype in rodent is more, and the Ces2 in mouse comprises 8 subtypes (Ces2a-Ces2h) which are located on a full-length 286kb gene cluster on chromosome 8. The Ces2 also includes 8 subtypes in rats and are located on chromosome 1 and chromosome 19, respectively. In rats, Ces2c and Ces2i are located on chromosome 1, and Ces2a, Ces2e, Ces2g, Ces2h, Ces2j and Ces2(AB010632) are located on chromosome 19. The Ces2 subtypes with higher expression in the liver and intestinal tract of rats and mice are reported to be Ces2c, Ces2a and Ces2e in sequence. At present, no research is carried out to determine that rodents contain the orthologous gene of human CES2, but the gene structures of multiple gene families of the rodents and the human CES are similar to each other and form a CES2-CES3A-CES4A structure, and the substrates and functions of the Ces2 in the humans and the rodents have high similarity, so that the orthologous gene of the human CES2 is commonly used as an important tool for researching the Ces 2. Compared with a mouse, a rat has a large body size, is easy to grab, has a large blood volume, can continuously take blood for multiple times and is commonly used for pharmacokinetic research. However, due to the complexity of the Ces2 gene in rats and mice, no animal model for knockout of the Ces2 gene has been constructed for drug metabolism and lipid metabolism related studies. The establishment of the Ces2 gene knockout rat model can more scientifically and effectively research the function of Ces2 and provide a powerful tool for drug metabolism research.
Disclosure of Invention
The invention utilizes the CRISPR/Cas9 system to target Ces2a and Ces2c in rats, successfully constructs a Ces2a/j, Ces2c and Ces2a/c/j gene knockout rat model for the first time, provides a brand new animal model for researching drug metabolism based on Ces2, and also provides a powerful tool for researching the functions of each Ces2 subtype in rats. The established Ces2a/j, Ces2c and Ces2a/c/j gene knockout rat model can be used for researching the drug metabolism function of each subtype of Ces2 in a rat, and provides a certain reference basis for more scientifically and accurately extrapolating the preclinical research results in the rat to a human. In addition, the model can be used for researching the role of the Ces2 in lipid metabolism and the relation between the Ces2 and the occurrence and development of diseases in rats, and provides some references for clinically treating metabolic diseases.
In order to research the metabolic function of Carboxylesterase 2(Ces2) and provide a brand-new animal model for drug metabolism research, the invention firstly uses a CRISPR/Cas9 system to realize the knockout of Ces2a/j, Ces2c and Ces2a/c/j genes in a rat body, and prepares the rat liver microsome with a specific Ces2 gene knockout. According to the invention, a specific Ces2 gene knockout rat model is successfully obtained by using a CRISPR/Cas9 system through the steps of target selection, sgRNA synthesis, embryo microinjection, rat feeding, propagation and the like, and a specific Ces2 gene knockout rat liver microsome solution is obtained through the steps of liver extraction, homogenization, differential centrifugation and the like, and can be used for drug metabolism related research.
The invention provides a method for constructing a Ces2 gene knockout rat model, which comprises the following steps:
1) determination of knockout targets
The CRISPR/Cas9 system knockout target was selected using the CRISPR design tool (on-line design tool http:// tools. genome-engineering. org), i.e. the knockout target was two 18bpDNA sequences ending with NGG. The knock-out target was chosen as close to the 5' end of the genome as possible.
The target point for knockout is two 18bp DNA sequences which are separated by 50bp and end by NGG.
The knockout targets are as follows:
ces2 a: TTGGCTAGACTTCCTGGT (SEQ ID NO.1) and TCTCCTCCAGCATGTGCA (SEQ ID NO. 2);
ces2 c: CGGAAACAACCACATAGC (SEQ ID NO.3) and TATTCTTATCCATGTGTG (SEQ ID NO. 4);
2) and (3) in-vitro synthesis and transcription of the sgRNA double-stranded template.
In vitro transcription of the sgRNA double-stranded template was initiated by the T7 promoter. Firstly, taking an oligonucleotide chain (Oligo) which is synthesized in vitro and has a T7 promoter sequence and an 18bp target sequence and has a length of 60bp as a template, and carrying out PCR reaction by using a high-fidelity KOD enzyme to obtain a sgRNA double-chain template.
The oligonucleotide sequence (Oligo) comprises:
Ces2a sgRNA：
Oligo1:
GATCACTAATACGACTCACTATAGGGCCTTTGGCTAGACTTCCGTTTTAGAGCTAGAAAT(SEQ IDNO.5)
Oligo2:
GATCACTAATACGACTCACTATAGGTCTCCTCCAGCATGTGCAGTTTTAGAGCTAGAAAT(SEQ IDNO.6)
Ces2csgRNA：
Oligo1:
GATCACTAATACGACTCACTATAGGCGGAAACAACCACATAGCGTTTTAGAGCTAGAAAT(SEQ IDNO.7)
Oligo2:
GATCACTAATACGACTCACTATAGGTATTCTTATCCATGTGTGGTTTTAGAGCTAGAAAT(SEQ IDNO.8)
the sgRNA double-stranded template comprises:
Ces2a sgRNA
template 1:
GATCACTAATACGACTCACTATAGGTTGGCTAGACTTCCTGGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.9)
template 2:
GATCACTAATACGACTCACTATAGGTCTCCTCCAGCATGTGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.10)
Ces2c sgRNA
template 1:
GATCACTAATACGACTCACTATAGGCGGAAACAACCACATAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.11)
template 2:
GATCACTAATACGACTCACTATAGGTATTCTTATCCATGTGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.12)
performing transcription reaction by using a T7 transcription kit, extracting the sgRNA obtained by transcription by using a phenol chloroform method, and storing the extracted sgRNA at-80 ℃ for later use.
3) sgRNA and Cas9 mRNA microinjection
Preparing a pseudopregnant mother mouse, carrying out in-vitro microinjection and transplantation of a single-cell embryo, and breeding the rat.
The sgRNA and Cas9 mRNA were co-injected into the fertilized egg cytoplasm, and well-conditioned fertilized eggs were transplanted into the oviduct of a pseudopregnant female mouse.
The sgRNA injection concentration is 20-40 ng/mu L; preferably, it is 25 ng/. mu.L.
The Cas9 mRNA injection concentration is 30-60 ng/. mu.L; preferably, it is 50 ng/. mu.L.
The method comprises the following specific steps: the superovulation treatment is firstly carried out on a normal adult female mouse, and then the female mouse and a normal male mouse are in coop to ensure that the female mouse and the normal male mouse are normally mated and fertilized. Female mice that had been successfully mated were then sacrificed, and fertilized eggs were removed and cultured in vitro for 2 hours. And co-injecting sgRNA and Cas9 mRNA into fertilized egg cytoplasm by using a microinjection technology, wherein the sgRNA injection concentration is 25 ng/mu L, and the Cas9 mRNA injection concentration is 50 ng/mu L. Culturing for several hours, judging the survival state of the fertilized eggs through morphological observation, transplanting the survived fertilized eggs into the oviduct of a pseudopregnant female mouse, and allowing the fertilized eggs to naturally nidate, divide and develop.
4) Ces2 gene knockout rat model construction
Breeding a pseudopregnant mother rat to obtain an F0 generation single-gene or multi-gene heterozygote rat, breeding an F0 generation single-gene or multi-gene heterozygote rat and a wild type rat in a cage to obtain an F1 generation single-gene or multi-gene heterozygote rat, breeding an F1 generation rat with the same genotype in a cage to obtain a homozygote mutant rat, namely an F2 generation rat, namely the animal model of the Cer 2 gene homozygote knockout rat.
The step 4) also comprises the step of identifying the genotype of a Ces2 gene knockout rat: extracting genome DNA of a rat model, designing primer pairs according to upstream and downstream sequences of a target spot by taking the genome DNA as a template to perform PCR reaction, performing sequencing analysis on PCR products, and selecting rats subjected to non-triplex multiple gene editing at a single site to perform subsequent breeding according to the sequencing analysis result.
In one embodiment, the step of genotyping an F0 generation monogenic or polygenic heterozygous rat: cutting toes of F0-generation young mice when the young mice are 4-7 days old, extracting genome DNA, using the genome DNA as a template, designing primers according to upstream and downstream sequences of a target spot to perform PCR reaction, performing sequencing analysis on PCR products, and selecting rats subjected to non-triplex multiple gene editing at a single site to perform subsequent breeding according to the sequencing analysis result.
The primer pairs used in the PCR are as follows:
Ces2a
upstream: CTGCTGGCTATTGGCTTCC (SEQ ID NO.13)
Downstream: CCTGCTGCTTTCCATCCC (SEQ ID NO.14)
Ces2c
Upstream: AACTTTCACCCACGACAT (SEQ ID NO.15)
Downstream: AAGGTTACCATCAGTGCC (SEQ ID NO.16)
The Ces2 gene knockout provided by the invention comprises knockout of one or more Ces2 genes.
The invention also provides a Ces2 gene knockout rat model obtained by the construction method.
Wherein, the Ces2 gene knockout rat model comprises a Ces2 single gene knockout rat model and a Ces2 multiple gene knockout rat model.
The invention also provides application of the Ces2 gene knockout rat model in preparation of Ces2 gene deletion rat liver microsomes.
The invention provides a method for preparing Ces2 single-gene or multi-gene deletion rat liver microsome by using a Ces2 single-gene or multi-gene knockout rat model, wherein single or multiple gene function deletions occur in the liver microsome.
The invention also provides a preparation method of the liver microsome of the rats with the Ces2 gene deletion, CO2And (3) after the animal model of the Ces2 homozygote gene knockout rat is killed by suffocation, taking the liver of the Ces2 gene knockout rat, and obtaining the liver microsome of the Ces2 gene deletion rat through homogenization and differential centrifugation.
The invention also provides the mouse liver microsome with the Ces2 gene deleted, which is prepared by the method.
The Cer 2 gene-deleted rat liver microsomes comprise a Cer 2 single-gene-deleted rat liver microsomes and a Cer 2 multi-gene-deleted rat liver microsomes.
The invention also provides a method for breeding a Ces2 multi-subtype knockout rat, which comprises the steps of mating two Ces2 single-gene or multi-gene homozygous knockout rats in cages to obtain a double-heterozygote or multi-heterozygote progeny rat, and selfing the double-heterozygote or multi-heterozygote progeny rats with the same nest and different sexes to obtain a progeny homozygous knockout rat, wherein the progeny homozygous knockout rat comprises the Ces2 multi-gene knockout rat.
The 'Ces 2 single-gene or multi-gene homozygous knockout rat coop mating' comprises the step of mating Ces2 single-gene and Ces2 single-gene homozygous knockout rat coop, the step of mating Ces2 multi-gene and Ces2 multi-gene homozygous knockout rat coop, and the step of mating Ces2 single-gene and Ces2 multi-gene homozygous knockout rat coop.
The self-crossing of the rats with different litters and different sexes by the double heterozygote or multi-heterozygote offspring comprises the self-crossing of the rats with different litters and different sexes by the double heterozygote and the double heterozygote offspring and the self-crossing of the rats with different litters and different sexes by the multi-heterozygote and the multi-heterozygote offspring.
In a specific embodiment, taking breeding of a Ces2a/c/j multi-subtype knockout rat as an example, a Ces2a/j homozygous knockout rat and a Ces2c homozygous knockout rat of different sexes are bred in a cage for more than 8 weeks to generate a three-gene heterozygous F1 generation rat, and an F2 generation containing the three-gene homozygous knockout rat is bred in a cage for the F1 generation heterozygous rat of the same litter of different sexes.
The invention obtains a Ces2 single-gene homozygous knockout rat model or a multi-gene knockout rat model positioned on the same chromosome by a 'construction method of a Ces2 gene knockout rat model', and obtains a Ces2 multi-subtype knockout rat model positioned on different chromosomes by a 'cultivation method of a multi-subtype Ces2 gene knockout rat model'. And the Ces2 multi-subtype knockout rat model located on different chromosomes is bred by a Ces2 single-gene homozygous knockout rat model by using the breeding method of the multi-subtype Ces2 gene knockout rat model.
The invention also provides a sgRNA double-strand template shown in any one of sequences SEQ ID NO.9-SEQ ID NO. 12.
Ces2a sgRNA
Template 1:
GATCACTAATACGACTCACTATAGGTTGGCTAGACTTCCTGGTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.9)
template 2:
GATCACTAATACGACTCACTATAGGTCTCCTCCAGCATGTGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.10)
Ces2c sgRNA
template 1:
GATCACTAATACGACTCACTATAGGCGGAAACAACCACATAGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.11)
template 2:
GATCACTAATACGACTCACTATAGGTATTCTTATCCATGTGTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT(SEQ ID NO.12)
the invention also provides application of the sgRNA in construction of a mouse model with a Ces2 gene knockout gene.
The invention also provides a primer pair as described above.
The invention also provides application of the primer pair in constructing a Ces2 gene knockout rat model.
The invention also provides application of the Ces2 gene knockout rat model or the Ces2 gene knockout rat liver microsome in vitro drug metabolism, drug toxicity evaluation and compound screening.
The invention also provides application of the Ces2 homozygous gene knockout rat model in drug metabolism research. To study drug metabolism, drug metabolizing enzymes involved in the metabolism need to be determined. For example, the metabolism of a test drug is detected in both a Ces2 knockout rat and a wild-type rat, and it can be determined whether the drug is metabolized by Ces2, and it can also be determined whether the generation of toxicity of the drug is mediated by Ces 2.
The invention also provides application of the Ces2 homozygous gene knockout rat model in liver lipid metabolism research. Liver lipid metabolism disorder in the Ces2 knockout rat can be found by simultaneously detecting liver structures of a wild rat and the Ces2 knockout rat. The rats with Ces2 gene knockout model can be used for exploring the causes of lipid metabolism disorder.
The technical difficulties to be overcome by the invention are as follows: ces2 subtypes are numerous, gene similarity among partial subtypes can reach 95%, other subtype interferences need to be eliminated when the expression of a single subtype is verified to be absent, and primer specificity is guaranteed when a primer is designed to ensure the specificity of amplification. Since the Ces2 subtypes are not on the same chromosome in rats, construction of a Ces2 multiple subtype knockout rat requires co-caging breeding of multiple single gene knockout rats.
The invention creatively constructs a Ces2 gene knockout rat model by using a CRISPR/Cas9 system for the first time, and prepares liver microsomes by using the rat model and uses the liver microsomes in the research of drug metabolism. The advantages of the invention include:
the Ces2 gene knockout rat model constructed by the CRISPR/Cas9 system has the advantages of short period, easy operation, high success rate and relatively low price.
The Ces2 knockout model is not reported in both rats and mice. Meanwhile, compared with a mouse, the rat has wider application prospect in the field of drug metabolism because the rat is large in body size, easy to grab and large in blood volume, and is suitable for continuously taking blood for multiple times.
Compared with the existing in-vitro inhibitor model, the rat liver microsome with the Ces2 gene deletion has stronger specificity and no toxicity, and has wide application prospect in-vitro evaluation of the metabolic pathway and toxicity of the compound.
In conclusion, the invention successfully knocks out a specific Ces2 subtype in a rat body and verifies the genotype and the metabolic function of the rat body. And the rat model is utilized to prepare liver microsomes with specific Ces2 subtype deletion, and the liver microsomes can be used for in vitro research on drug metabolism, drug toxicity evaluation and compound screening.
In a specific embodiment, the invention describes a detection result of a knocked-out rat serum physiological index, and proves that a Ces2 gene knocked-out rat model constructed by using a CRISPR/Cas9 system has no significant difference in physiological conditions compared with a wild rat, and the knocked-out rat can normally grow and reproduce, so that the growth, development and reproduction of the rat are not influenced by the knocking-out of Ces 2.
The Ces2 gene knockout rat model obtained by the invention has gene deletion, transcriptional level expression and metabolic function deletion. In the specific example, agarose gel electrophoresis of the PCR product of cDNA proves that the expression of Ces2a transcription level is lost, and the product yield of a knockout rat is far lower than that of a wild rat in an in vivo pharmacokinetics experiment of diltiazem which is a specific substrate of Ces2a, so that the loss of the Ces2a metabolic function is proved.
The enzyme activity deletion of the Ces2a of the liver microsome of the Ces2a/j knockout rat prepared by the invention is proved by in-vitro enzyme kinetic analysis of a specific substrate diltiazem. In a specific embodiment, the maximum reaction rate V of metabolism of diltiazem by Ces2a in microsome incubation of liver of a Ces2a/j knockout ratmaxAnd intrinsic clearance CLintAre much lower than wild type rats.
Drawings
FIG. 1 shows the identification results of the genotypes of the Ces2a and Ces2c knockout rat model F0. Panel A shows the identification of the F0 genotype of Ces2a knockout rats, and the arrows indicate rats with large fragment gene editing. Panel B shows the identification of the F0 genotype of the Ces2c knockout rat, and the arrow indicates the rat with large fragment gene editing.
FIG. 2 shows the result of genotyping identification and sequencing of the F1 generation of a Ces2a knockout rat model. Panel A is wild type and panel B is heterozygote.
FIG. 3 shows the identification results of F2 generation genotypes of Ces2a and Ces2c knockout rats. Panel A shows the identification result of the genotype of the F2 generation of a Ces2a knockout rat model. B is the result of genotype identification of the F2 generation of a Ces2c knockout rat model.
FIG. 4 shows the expression difference of Ces2a at the transcriptional level in the liver (A) and small intestine (B) of wild type rats and Ces2a/j knockout rats and Ces2a/c/j knockout rats.
FIG. 5 is a graph showing the in vitro enzyme kinetics curves and related parameters of diltiazem metabolism by liver microsomes prepared from Ces2a/j knockout rats and wild type rats.
FIG. 6 shows the pharmacokinetics (A) and area under the drug curve (AUC) (B) of diltiazem in Ces2a/j knockout rats and wild type rats.
FIG. 7 is a graph showing the metabolic capability of methylprednisolone succinate in wild type rats and Ces2a/c/j knockout rats.
FIG. 8 shows H & E staining results of liver of wild type rat and Ces2a/c/j knockout rat. The arrows indicate lipid vacuoles. The scale bar of the figure is 100 μm.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, the contents of which include but are not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.