NL2030466A - Construction method of conditional overexpression model of genetic locus-specific insertion gene - Google Patents
Construction method of conditional overexpression model of genetic locus-specific insertion gene Download PDFInfo
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Abstract
UITTREKSEL The present disclosure belongs to the technical field of animal model construction, and discloses a construction method of a conditional overeXpression model of a genetic locus—specific insertion gene (Rosa26—LSL—IE2). The construction method includes 5 the following steps: obtaining a Cas9 mRNA and a gRNA through in vitro transcription; constructing a homologous recombination vector, donor vector, by an In—Fusion cloning method; conducting Hdcroinjection on the Cas9 mRNA, the gRNA and the donor vector into oosperm of a C57BL/6J mouse to obtain a FO—generation mouse; 10 and allowing a positive FO—generation mouse identified by polymerase chain reaction (PCR) amplification and sequencing and the C57BL/6J' mouse to mate to obtain. a positive Fl—generation mouse. In the present disclosure, modeling of an IE2 transgenic mouse successfully overcomes species specificity to construct a 15 body environment capable of stably and continuously expressing IE2 protein. (+ Fig. l)
Description
TECHNICAL FIELD The present disclosure belongs to the technical field of ani- mal model construction, and in particular relates to a construc- tion method of a conditional overexpression model of a genetic lo- cus-specific insertion gene.
BACKGROUND ART At present, human cytomegalovirus (HCMV), belonging to a p subfamily of Herpesviridae, is a linear double-stranded DNA virus. The HCMV with biological characteristics of latent activation has an infection rate in the population up to 100%. Normal people in- fected with HCMV show asymptomatic recessive or latent infection. However, once the human immune function undergoes major changes, such as tumors, HIV infection, organ transplantation and newborns, the HCMV can be activated from the latent infection to cause pri- mary and secondary proliferative infections, leading to severe clinical symptoms or even death. Congenital and perinatal viral infections are main viral causes of birth defects, mainly causing congenital malformations of the central nervous system (CNS). Neu- ral stem cells, neurons and glial cells are generally susceptible to the HCMV, such that embryonic HCMV infection always leads to severe CNS malformations. Only 10-15% of fetuses have obvious symptoms after being infected with the HCMV. However, over 90% of babies may be born asymptomatically with latent infections, and will gradually develop into delayed nervous system damages such as deafness and mental retardation after a few years. Due to species specificity, a molecular mechanism of the HCMV has not been eluci- dated so far in long-term persistent infection in the body and in a relationship between diseases. Therefore, there is an urgent need for an animal model that can effectively clarify the molecu- lar mechanism of the HCMV in long-term persistent infection in the body and in the relationship between diseases.
Through the above analysis, the prior art has the problems and shortcomings as follows: due to the species specificity, the molecular mechanism has not been elucidated so far in long-term persistent infection of the HCMV in the body and in the relation- ship between the HCMV and diseases.
The difficulty of solving the above problems and shortcomings is that: due to the species specificity, previous studies are lim- ited to in vitro on the mechanism of neurological damages caused by the HCMV infection. The in vitro environment cannot completely simulate the in vivo environment, such that the research is lim- ited on the mechanism of congenital HCMV causing neurological dis- eases. A conditional overexpression model of a Rosa26-specific IE2 gene, abbreviated as: Rosa26-LSL-IE2, is constructed to better clarify the molecular mechanism of the nervous system damages caused by the congenital HCMV infection in the body.
The significance of solving the above problems and shortcom- ings is that: the modeling of this model successfully overcomes the species specificity of HCMV to construct a body environment capable of stably and continuously expressing ie2. This will help to further clarify the mechanism of congenital HCMV infection leading to neurological diseases and to provide a theoretical ba- sis for disease prevention and treatment.
SUMMARY In view of this, the present disclosure provides a construc- tion method of a conditional overexpression model of a genetic lo- cus-specific insertion gene.
The present disclosure provides a construction method of a conditional overexpression model of a genetic locus-specific in- sertion gene, including: inserting a CAG-LSL-IE2-WPRE-pA expres- sion cassette into a Rosa26 genetic locus using CRISPR/Cas9 through homologous recombination, to establish an JIE2 transgenic mouse model.
Further, a construction method of the Rosa26-LSL-IE2 model may include the following steps: step 1, obtaining a Cas9 mRNA and a gRNA through in vitro transcription (in preparation for positioning restriction sites and conducting accurate cleavage); step 2, constructing a homologous recombination vector, donor vector, by an In-Fusion cloning method (construction of an expres- sion vector); step 3, conducting microinjection on the Cas9 mRNA, the gRNA and the donor vector into oosperm of a C57BL/6J mouse to obtain a FO-generation mouse (insertion of a target gene); and step 4, allowing a positive FO-generation mouse identified by polymerase chain reaction (PCR) amplification and sequencing and the C57BL/6J mouse to mate to obtain a positive Fl-generation mouse Obtaining method (obtaining a transgenic positive mouse).
Further, in step 1, the gRNA may have a gene sequence as shown in SEQ ID NO: 1. Further, in step 2, the homologous recombination vector, vec- tor donor, may include a 3.3 kb 5'-homologous arm, a CAG-LSL-IE2- WPRE-pA and a 3.3 kb 3'-homologous arm.
Further, in step 3, the F0-generation mouse may be a chimera. Further, in step 4, genotype identification of the FO- generation mouse may include: (1) transplanting ocosperm after injection into a pseudo- pregnant female mouse, to obtain a FO-generation mouse born after 20 days; and (2) conducting PCR amplification and sequencing, and genotype identification.
The present disclosure further provides a conditional overex- pression model of a genetic locus-specific insertion gene con- structed by the construction method of the Rosa26-LSL-TIE2 model, where the conditional overexpression model of a genetic locus- specific insertion gene is an IE2 transgenic mouse model.
The present disclosure further provides use of the condition- al overexpression model of a genetic locus-specific insertion gene in detection of long-term persistent infection of an HCMV in a body.
The present disclosure further provides use of the condition- al overexpression model of a genetic locus-specific insertion gene in an animal model of a molecular mechanism of a relationship be- tween the HCMV and a disease.
The present disclosure further provides a construction method of an ie2 for sustained expression, using the conditional overex- pression model of a genetic locus-specific insertion gene.
Combining all the above technical solutions, the present dis- closure have the advantages and positive effects as follows: an IE2 transgenic mouse model is established by the construction method of a conditional overexpression model of a genetic locus- specific insertion gene provided by the present disclosure; the modeling of the IE2 transgenic mouse model successfully overcomes the species specificity to construct a body environment capable of stably and continuously expressing ie2. This will help to further clarify the mechanism of the ie2 leading to neurological diseases and to provide a theoretical basis for disease prevention and treatment.
BRIEF DESCRIPTION OF THE DRAWINGS To describe the technical solutions in embodiments of the present disclosure more clearly, the following briefly describes the accompanying drawings that need to be used in the embodiments.
Apparently, the accompanying drawings in the following description show merely some embodiments of the present disclosure, and a per- son of ordinary skill in the art may derive other drawings from these accompanying drawings without creative efforts.
FIG. 1 shows a flow chart of a construction method of a con- ditional overexpression model of a genetic locus-specific inser- tion gene provided by an example of the present disclosure; FIG. 2 shows a schematic diagram of a mouse construction strategy provided by an example of the present disclosure; FIG. 3 shows a plasmid map of a homologous recombination vec- tor provided by an example of the present disclosure; FIG. 4 shows an electrophoresis diagram of restriction diges- tion identification of the homologous recombination vector provid- ed by an example of the present disclosure; where S: identification results of SacII restriction analysis, with a theoretical band size of 10,127 bp, 3,663 bp, 2,561 bp, 1,635 bp and 528 bp; and M: a 1 kb DNA ladder; FIG. 5 shows a schematic diagram of specific expression of ie2 in a hippocampus of a offspring by mating this model mouse with a CamkII-Cre mouse provided by an example of the present dis- closure; and FIG. 6 shows a schematic diagram of a behavioral experiment 5 proving that expression of the ie2 can cause abnormal development of a mouse nervous system provided by an example of the present disclosure.
DETAILED DESCRIPTION OF THE EMBODIMENTS To make the objective, technical solutions and advantages of the present disclosure clearer and more comprehensible, the pre- sent disclosure will be further described below in detail in con- junction with embodiments. It should be understood that the spe- cific examples described herein are merely intended to explain the present disclosure, rather than to limit the present disclosure.
In view of this, the present disclosure provides a construc- tion method of a conditional overexpression model of a genetic lo- cus-specific insertion gene. The present disclosure will be de- scribed in detail below in conjunction with the accompanying draw- ings.
An example of the present disclosure provides a construction method of a conditional overexpression model of a genetic locus- specific insertion gene, including: inserting a CAG-LSL-IE2-WPRE- PA expression cassette into a Rosa26 genetic locus using CRISPR/Cas9 through homologous recombination, to establish an IE2 transgenic mouse model.
As shown in FIG. 1, the construction methed of a conditional overexpression model of a genetic locus-specific insertion gene includes the following steps: 8101, obtaining a Cas9 mRNA and a gRNA through in vitro tran- scription; 5102, constructing a homologous recombination vector, donor vector, by an In-Fusion cloning method; 3103, conducting microinjection on the Cas9 mRNA, the gRNA and the donor vector into oosperm of a C57BL/6J mouse to obtain a FO-generation mouse; and 3104, allowing a positive FO0-generation mouse identified by
PCR amplification and sequencing and the C57BL/6J mouse to mate to obtain a positive Fl-generation mouse. The technical solutions of the present disclosure are further described below in conjunction with the embodiments.
In the present disclosure, to have a more comprehensive and accurate understanding of some pathogenic mechanisms of the HCMV in a host, an IEZ transgenic mouse model is established. The mod- eling of IE2 transgenic mouse successfully overcomes the species specificity to construct a body environment capable of stably and continuously expressing ie2. This will help to further clarify the mechanism of the ie2 leading to neurological diseases and to pro- vide a theoretical basis for disease prevention and treatment.
In the present disclosure, the CAG-LSL-IE2-WPRE-pA expression cassette is inserted into the Rosa26 genetic locus using CRISPR/Cas9 through homologous recombination. A brief process is as follows: the Cas9 mRNA and the gRNA are obtained through in vitro transcription; the homologous recombination vector, donor vector, is constructed by the In-Fusion cloning method. This vec- tor includes a 3.3 kb 5'-homologous arm, a CAG-LSL-IE2-WPRE-pA and a 3.3 kb 3'-homologous arm. The microinjection was conducted on the Cas9 mRNA, the gRNA and the donor vector into the oosperm of the C57BL/6J mouse to obtain the FO-generation mouse. The positive FO-generation mouse identified by PCR amplification and sequencing and the C57BL/6J mouse are allowed to mate to obtain the positive Fl-generation mouse.
1. Design strategy
1.1. The schematic diagram of the design strategy is shown in FIG. 2.
1.2. Sequence information of gRNA (Table 1) The gRNA has a gene sequence as shown in SEQ ID NO: 1. Table 1 Sequence information of gRNA
2. Construction of homologous recombination plasmid
2.1. The map of the homologous recombination plasmid is shown in FIG. 3 and Table 2. Table 2 Description of map of homologous recombination plas- mid == CAG promoter CAG promoter (a strong promoter expressed in mammalian cells) Polyadenylic acid tailing signal WPRE Woodchuck hepatitis virus post- transcriptional regulatory element (WPRE; for enhancing stability of RNA transcrip- tion) 3'-homologous arm
3.2. The identification by restriction analysis of homologous recombination plasmid is shown in FIG. 4.
3.3. Genotype identification of the FO-generation mouse The oosperm after injection is transplanted into the pseudo- pregnant female mouse, to obtain the FO-generation mouse born af- ter 20 days. The PCR amplification and sequencing are conducted for genotype identification. Since the oosperm has a high early cleavage rate, the FO0-generation mouse obtained is a chimera that may not have stable inheritance. Passage is required to obtain a Fl-generation mouse with the stable inheritance. The technical effects of the present disclosure will be de- scribed in detail below in conjunction with the experiments. Insertion site gene (Ensembl) has a name of: Gt (ROSA) 26Sor (ENSMUSGQO0000086429), abbreviated as: Rosa26. A URL link of the insertion site gene Ensembl is: http://asia.ensembl.org/Mus musculus/Gene/Summary?db=core;g=E NSMUSGO0000086429;r=6:113044389-113054294. Insertion site chromosome position (Ensembl) is: Chromo- some6:113076031. IE2 gene sequence is: https: //www.ncbi.nlm.nih.gov/nuccore/NC 006273.2?location=174 020:174090:2,173719:173903:2,170689:172175:2&report=fasta.
As shown in FIG. 5, there is specific expression of ie2 in a hippocampus of a offspring by mating this model mouse with a CamkII-Cre mouse (Western-bolt; the ie2 protein has a size of 86 KD) .
As shown in FIG. 6, a behavioral experiment proves that ex- pression of the ie2 can cause abnormal development of a mouse nervous system.
The foregoing are merely descriptions of the specific embodi- ments of the present disclosure, and the protection scope of the present disclosure is not limited thereto. Any modification, equivalent replacement, improvement, etc. made within the tech- nical scope of the present disclosure by a person skilled in the art according to the spirit and principle of the present disclo- sure shall fall within the protection scope of the present disclo- sure.
Sequence Listing <110> Qingdao University Qingdao WanMing BioCell Pharmaceutics Co., Ltd <120> CONSTRUCTION METHOD OF CONDITIONAL OVEREXPRESSION MODEL OF GENETIC LOCUS-SPECIFIC INSERTION GENE <130> HKJP202111933 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the gRNA <400> 1 ggggacacac taagggagct tgg 23
SEQUENCE LISTING <110> Qingdao University Qingdao WanMing BioCell Pharmaceutics Co., Ltd <120> CONSTRUCTION METHOD OF CONDITIONAL OVEREXPRESSION MODEL OF GENETIC LOCUS-SPECIFIC INSERTION GENE <130> HKJP202111933 <160> 1 <170> PatentIn version 3.5 <21e> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the gRNA <400> 1 ggggacacac taagggagct tgg 23
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