CN113564203A - Preparation method and application of HSV1-tk/GCV induced blood system defect mouse model - Google Patents
Preparation method and application of HSV1-tk/GCV induced blood system defect mouse model Download PDFInfo
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- CN113564203A CN113564203A CN202110753676.8A CN202110753676A CN113564203A CN 113564203 A CN113564203 A CN 113564203A CN 202110753676 A CN202110753676 A CN 202110753676A CN 113564203 A CN113564203 A CN 113564203A
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Abstract
The invention provides a mouse model of transduction HSV1-tk suicide gene with high expression in blood system specificity and a preparation method and application thereof, the transgenic mouse HS21/45vav1-HSV1-tkB6/C57 model of the invention eliminates a receptor immune system in allogeneic and xenogeneic organ transplantation, has great application value in avoiding rejection reaction, realizes the construction of a chimera of the allogeneic blood system, finally realizes the chimerism of the xenogeneic blood system, creates conditions for transplanting allogeneic or xenogeneic hematopoietic stem cells to the receptor B6/C57 mouse, can also be applied to large animal models which are difficult to be irradiated, such as pigs and non-human primates, and simultaneously has profound research significance in the immune tolerance aspect in xenogeneic organ reconstruction.
Description
Technical Field
The invention relates to a preparation method and application of a mouse model, in particular to an animal model which is induced by a hematopoietic system specific gene Vav1 promoter and has high specificity expression of HSV1-tk in a blood system of a suicide gene-herpes simplex virus 1 type thymidine kinase (HSV1-tk) transgenic B6/C57 mouse, and induces the defect of the blood system through medicine GCV, and a preparation method and application thereof.
Background
Blood system chimerism is a robust and reliable method for studying the immunogenicity and immune tolerance of allogeneic or xenogeneic transplanted donors. Studies have demonstrated that human embryo thymus, liver and CD34 were co-transplanted+Stem cells to severe combined immunodeficiency (NOD/SCID) mice can reconstruct a functional human immune system in NOD/SCID mice to obtain humanized mice of the immune system, which indicates that it is feasible to reconstruct the human immune system in mice, and now, humanized mouse models become important model tools in the fields of human hematopoietic function, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, regenerative medicine and the like. However, the humanized mice at the present stage are prepared based on severe combined immunodeficiency mice, and have certain limitations in research application, and have the defects of high requirement on feeding conditions, short survival time and the like in the later application process. To date, reconstitution of the human immune system has not been achieved in normal B6/C57 mice.
Disclosure of Invention
The invention aims to solve the technical problem of providing a mouse model for transducing HSV1-tk suicide gene to be specifically and highly expressed in a blood system and a preparation method and application thereof.A transgene constructing vector is inserted into an expression plasmid of HSV1-tk driven by a Vav1 promoter, a transgene vector is linearized, and after purification, nucleic acid with a target gene is injected into B6/C57 mouse fertilized eggs by a microinjection technology, so that an exogenous gene is integrated into a mouse genome, and the effect efficiency of the exogenous gene on killing and removing the blood system and the influence on functions of later-stage heterogenous hematopoietic stem cell chimeric body formation and the like are detected.
In order to solve the technical problems, the invention adopts the technical scheme that:
the recombinant vector expression unit is characterized by comprising a Vav1 promoter, three structural domains of HS2/1, HS4 and HS5 and a suicide gene HSV 1-tk.
Furthermore, the expression unit of the recombinant vector consists of a Vav1 promoter-HS 2/1, a suicide gene HSV1-tk, a Vav1 promoter-HS 4 and a Vav1 promoter-HS 5 which are connected in sequence.
Further, the recombinant vector expression unit is constructed as follows: HS2/1, HS4 and HS5 structural domains of Vav1 gene and suicide gene HSV1-tk are amplified, and the suicide gene HSV1-tk is inserted between Vav1 promoter-HS 2/1 and Vav1 promoter-HS 4 according to the sequence of Vav1 promoter-HS 2/1, Vav1 promoter-HS 4 and Vav1 promoter-HS 5.
More preferably, the nucleotide sequences of the Vav1 promoter-HS 2/1, the suicide gene HSV1-tk, the Vav1 promoter-HS 4 and the Vav1 promoter-HS 5 are respectively shown in SEQ ID NO. 1-4.
The invention also claims a vector containing the expression unit of any one of the recombinant vectors.
As another technical solution of the present invention,
the invention also relates to a construction method of the transgenic animal model, which is characterized by comprising the following steps: integrating the vector of claim 5 into the genome of an animal.
Specifically, the construction method specifically comprises the following steps: extracting and purifying the carrier, performing linearization treatment, integrating a target gene into an animal genome by utilizing fertilized egg prokaryotic injection, transplanting an oviduct embryo into a surrogate pregnant mouse, obtaining a first-built mouse after 21 days, cutting off 2 mm rat tail when a newborn mouse grows to two weeks, extracting a rat tail genome by an alkaline cracking method, designing HSV1-tk specific primers, performing genotype identification, and taking a positive first-built mouse with an HSV1-tk target band.
As another technical solution of the present invention,
the invention also relates to a method for preparing the chimeric animal model, which is characterized by comprising the step of transplanting the whole bone marrow of the obtained positive initial mouse to a wild normal mouse which is subjected to irradiation treatment.
As another technical solution of the present invention,
the invention also relates to the application of the animal model in xenogeneic organ transplantation, xenogeneic blood system fusion, recipient bone marrow elimination, xenogeneic organ reconstruction or immune tolerance mechanism research.
The invention also relates to the use of the animal model as a model animal for the study of the cellular function and phenotype of the hematopoietic and immune systems.
As another technical solution of the present invention,
a method for preparing a mouse model for transducing HSV1-tk suicide gene to have high specificity expression in a blood system comprises the following steps:
1) vector construction: from pORF-HSV1-tk, HSV1-tk was PCR amplified, introducing the required NotI and SfiI cleavage sites: the upstream primer is 5'-GCAGGCCCGATCGGCCATGGCCTCGTACCCCGGC-3', and the introduced SfiI restriction enzyme site is underlined; the downstream primer is 5'-GCATCAGCGCGGCCGCTCAGTTAGCCTCCCCCAT-3', and the introduced NotI enzyme cutting site is underlined; the reaction condition is 94 ℃, 5min pre-denaturation; the following cycle was performed 30 times: 10s at 98 ℃, 30s at 62 ℃ and 1min at 68 ℃; then final extension is carried out at 72 ℃ for 5 min; and finally, storing at 4 ℃, amplifying the fragment at 1130bps, and separating by electrophoresis in 1% agarose gel after PCR amplification is finished, wherein the conditions are as follows: the voltage is 120V, and the time is 25 minutes; after confirming the size of the band under an ultraviolet lamp, cutting off the target band with a clean blade, recovering and purifying the target fragment DNA with glue, detecting the concentration of the recovered DNA with a Nanodrop 2000, ligating a Blunt vector, competent transformation of the ligation product with Trasnt1, coating the ligation product on an ampicillin resistant plate, culturing the plate in a constant temperature incubator at 37 ℃ for 12 to 14 hours, selecting single clone in a super clean bench, carrying out SfiI and NotI double enzyme digestion verification after plasmid is extracted, carrying out amplification culture on the clone with correct sequencing, extracting plasmids, carrying out double enzyme digestion by SfiI and NotI to obtain an HSV1-tk gene fragment, recycling and purifying glue, measuring the concentration, carrying out double enzyme digestion purification on HS21/45vav1 by SfiI and NotI, carrying out T4 connection with the purified HSV1-tk after the concentration is measured, carrying out T4 connection for 1 hour at 25 ℃, and (3) transforming the competent cells of TransT1, culturing, selecting a single clone, and obtaining a positive clone after SfiI and NotI double enzyme digestion verification to obtain a transgenic plasmid: HS21/45vav1-HSV 1-tk;
2) linearization and purification of transgenic vectors: carrying out single enzyme digestion on the constructed HS21/45vav1-HSV1-tk plasmid by HindIII, removing a prokaryotic expression part in the vector, completing linearization, separating the plasmid subjected to enzyme digestion by 80V voltage, cutting glue, purifying QIAGENE DNA Kit and recovering a target fragment to obtain a purified transgenic vector HS21/45vav1-HSV 1-tk;
3) preparation of mouse model: injecting a transgenic vector HS21/45vav1-HSV1-tk into a male pronucleus of a fertilized egg of a C57 mouse, implanting the transgenic vector into a uterus of a pseudopregnant mouse, delivering the transgenic vector 19-21 days after surgery, cutting toes, numbers and tails after 3 weeks of newborn mice, extracting genome DNA of each mouse, detecting the integration condition of a target gene HSV1-tk by using a PCR technology, and further obtaining a first-construction mouse for transducing HS21/45vav1-HSV 1-tk;
4) establishing an HS21/45vav1-HSV1-tk transgenic mouse line: mating and passaging the initial mouse integrated with the exogenous target gene with a wild type normal C57 male mouse of 6-8 weeks old, extracting genome DNA of a mouse tail of a offspring, and identifying a positive baby mouse carrying HS21/45vav1-HSV1-tk by PCR.
5) GCV maximum safe dose exploration: wild-type normal B6/C57, 6-8 week old male and female mice were randomly assigned to PBS group, GCV treated group, 50 mice each: five groups of 20mg/kg, 40mg/kg, 80mg/kg and 160mg/kg are treated for 2 weeks, the weight of the mice is measured every three days in the treatment period, the state of the mice is observed, the dead mice are recorded, the mice are killed after 2 weeks, the survival conditions of peripheral blood and bone marrow cells of the mice of each group are detected, the weight change of important organs of the mice of each group is weighed, and the histology observation is carried out on each tissue of the mice of each group; by combining all indexes, the mice have obvious toxic reaction when the GCV treatment concentration is 160mg/kg, so that the GCV treatment concentration of later in-vivo experiments is designed to be between 80mg/kg and 100 mg/kg.
6) Obtaining of allogeneic blood system chimera: transplanting the whole bone marrow of a positive F1 generation HS21/45vav1-HSV1-tk-C57(CD45.2) mouse into a wild type normal B6/C57(CD45.1/2) irradiated by myeloablative, after 4 weeks, analyzing the distribution of cells of each lineage in peripheral blood by flow analysis to know that HS21/45vav1-HSV1-tk positive cells (CD45.2) have successfully replaced original cells (CD45.1/2), after 2 weeks of GCV treatment, carrying out chimeric replacement by using bone marrow cells of a wild type normal B6/C57(CD45.1) mouse, continuously carrying out GCV treatment for 4 weeks, simultaneously carrying out serial transplantation once at an interval of one week, after 4 weeks of GCV treatment, stopping treatment for 4 weeks, killing the mouse, carrying out flow detection on peripheral blood of the mouse in each group and taking up the chimeric replacement cells in the bone marrow, wherein the PBS treatment group is a control group, peripheral blood is detected once a week by flow detection, and long-term observation results are combined, so that the receptor bone marrow cells can be well eliminated by treating mice with 80mg/kg GCV dose for 4 weeks before and after chimeric replacement transplantation, and allogeneic hematopoietic chimerism is realized.
The advantages achieved by the present invention mainly include, but are not limited to, the following:
the transgenic mouse HS21/45vav1-HSV1-tkB6/C57 model has great application value in clearing the immune system of a receptor in allogeneic and xenogeneic organ transplantation and avoiding rejection. In the invention, an HSV1-tk/GCV inducible killing system is combined with a blood system specific expression gene promoter to obtain a mouse model which can transduce a suicide gene, namely herpes simplex virus 1 thymidine kinase (HSV1-tk), and has high blood system specific expression, a non-radiative, safe and specific drug is used for inducing and killing a receptor B6/C57 mouse blood system to kill and block the generation of new T cells, and the effect of removing receptor hematopoietic stem cells is achieved, so that the physiological condition that donor hematopoietic stem cells enter receptor bone marrow is realized, an immune barrier and a physiological barrier are removed, a condition is created for transplanting allogeneic or xenogeneic hematopoietic stem cells to a receptor B6/C57 mouse, the construction of a chimera of the same blood system is realized, and the chimera of the xenogeneic blood system is finally realized. And the application of the invention in large animal models which are difficult to be irradiated and the research significance of immune tolerance in xenogeneic organ reconstruction are profound, such as pigs, non-human primates, and the like.
Drawings
FIG. 1 is a construction design diagram of HS21/45vav1-HSV 1-tk.
FIG. 2 shows the PCR results of each functional domain of Vav1 and the results of the enzyme-cleaved electrophoresis analysis of the backbone vector (nucleic acid molecular weight standard: DL15000 Marker).
FIG. 3 shows the electrophoresis diagram of HSV1-tk fragment after PCR (nucleic acid molecular weight standard: DL15000 Marker).
FIG. 4 shows the electrophoresis diagram of the single enzyme digestion linearization of HS21/45vav1-HSV1-tk transgenic vector HindIII (nucleic acid molecular weight standard: DL15000 Marker).
FIG. 5 shows the PCR identification of a Positive initial mouse, genotype identification electrophoretogram (nucleic acid molecular weight standard: DL15000Marker, number of newborn mouse 1, 2, 3, 4, 5, 6, and Positive is diluted transgenic plasmid).
FIG. 6 shows that PBMC of an HS21/45vav1-HSV1-tk positive newborn mouse is cultured in vitro, and after GCV treatment, the in vitro effectiveness of a killing system is verified by a CCK8 color development method.
FIG. 7 shows the in vitro culture of HS21/45vav1-HSV1-tk positive pup PBMC, and the flow analysis of CD3+ cell viability after GCV treatment (NO.16, NO. W, HS21/45vav1-HSV1-tk positive pup PBMC, WT, wild-type normal C57 mouse PBMC).
FIG. 8 shows the results of the detection of the expression level of apoptosis-related protein clear-caspase 3 protein and its gray scale analysis after GCV treatment in vitro cultured PBMC of HS21/45vav1-HSV1-tk positive pup mice (WT-0, WT-20 are wild-type normal C57 mouse PBMC, W-0, W-20, W-40, W-80 are HS21/45vav1-HSV1-tk positive pup mice PBMC.).
FIG. 9 is a design diagram of an experiment for verifying the effectiveness of the killing system in HS21/45vav1-HSV1-tk positive newborn mice in vivo.
FIG. 10 is an electrophoretogram (nucleic acid molecular weight standard: DL15000Marker, PC is a diluted transgenic plasmid.) showing the presence of HS21/45vav1-HSV1-tk verified by PCR of DNAs of GCV80mg/kg treatment group and solvent treatment control group in an experiment for verifying the effectiveness of a killing system in HS21/45vav1-HSV1-tk positive newborn mice in vivo.
Fig. 11 shows the weight monitoring results of the mice in each group in the GCV maximum safe dose exploration experiment, wherein the abscissa represents the time point, the ordinate represents the weight, and each color indicates the weight change of the mice in different groups.
Fig. 12 is a statistical result of the weights of the organs of the mice in each group after two weeks of treatment in the GCV maximum safe administration dose exploration experiment, wherein the abscissa is the tissue type, the ordinate is the weight, and the histogram in each tissue type indicates that the administration doses of the mice are, from left to right: PBS, 20mg/kg, 40mg/kg, 80mg/kg, 160 mg/kg.
FIG. 13 is a representative flow analysis graph of the flow analysis of the correlation between the survival rates of leukocytes in peripheral blood and bone marrow in a GCV maximum safe dose discovery experiment, from top to bottom: the first column is a single stained sample of CD45, the second is an isotype control stain of CD45 antibody, the third is a positive control stain of H2O2 treated, and the last is a representative sample set.
Fig. 14 is a statistical chart of flow analysis results regarding the survival rate of leukocytes in peripheral blood and bone marrow by flow analysis in the GCV maximum safe dose exploration experiment, with the abscissa being time points and the ordinate being the survival rate of leukocytes of mice in the experimental group relative to the control group, and histograms at each time point, indicating from left to right the mouse dose: PBS, 20mg/kg, 40mg/kg, 80mg/kg, 160 mg/kg.
Fig. 15 is a schematic diagram of flow analysis after chimeric replacement transplantation in the experiment for obtaining allogeneic blood system chimeras, in which the proportion of CD45.1 and CD45.2 positive cells and the proportion of T cells, B cells and myeloid cells in CD45.1 and CD45.2 were mainly analyzed.
FIG. 16 is a statistical graph of the change in absolute number of individual lineages in CD45.1, CD45.2 after 7 monitoring cycles of flow analysis after chimeric replacement transplantation in experiments with allogeneic blood system chimera, 80-M-110, 80-M-111, 40-M-113 are representative of three experimental groups of mice, with cell types of individual lineages on the ordinate and absolute cell numbers on the abscissa.
FIG. 17 is a representative flow analysis graph of the ratio of CD45.1 positive cells in hematopoietic stem cells in bone marrow cells measured from sacrificed mice 4 weeks after cessation of GCV treatment following chimeric replacement transplantation in experiments for acquisition of allogeneic blood system chimeras.
FIG. 18 is a statistical chart of the results of the ratio of CD45.1 positive cells in hematopoietic stem cells in bone marrow cells measured from mice sacrificed 4 weeks after termination of GCV treatment after chimeric replacement transplantation in experiments for acquisition of allogeneic blood system chimeras.
Detailed Description
In the research, a CRISPR/Cas9 gene editing technology is combined with an HSV1-tk/GCV system to construct a blood system specific expression vector, and a Ganciclovir (GCV) -induced blood system deficiency mouse model is further constructed, namely an HSV1-tk expression vector (Vav1 promoter-HSV1-tk) guided by a Vav1 promoter, firstly, an HS21/45Vav1-HSV1-tk transgenic mouse model is obtained by taking a B6/C57 mouse as a receptor, and a suicide system is used for removing the original blood system of the receptor, so that a host anti-graft reaction in later-stage xenogeneic organ transplantation is avoided.
The invention is described in further detail below with reference to the following figures and detailed description:
by analyzing the structure of the Vav1 gene, functional domains HS2/1, HS4 and HS5 determine the blood system specific expression of the gene, and then the vector structure is designed as shown in FIG. 1: the suicide gene HSV1-tk is inserted between HS2/1 and the functional domains HS4 and HS5, and finally the purpose of specifically expressing the suicide gene HSV1-tk in a blood system is achieved.
Example 1 based on the discovery of specific related domains of Vav1 gene in hematopoietic system as HS2/1, HS4 and HS5, the objective gene HSV1-tk and promoter region of Vav1 gene specifically expressed in hematopoietic system are structurally designed, as shown in FIG. 1.
Example 2 backbone vectors HS2/1, 4, 5 and vav1 fragment PCR and SfiI and NotI double digestion linearization products electrophoresis results are shown in FIG. 2, and the specific implementation mode is: performing PCR amplification by using a high-fidelity KOD-Plus-Neo DNA polymerase and a mouse genome as a template to obtain each functional domain, connecting T4, performing TransT1 competent transformation, identifying positive clones, performing plasmid extraction, performing electrophoresis separation on 1% agarose gel at 120V for 25 minutes after SfiI and NotI double-enzyme digestion linearization at 37 ℃, then performing DNA gel recovery, and storing at-20 ℃ for later use after determining the concentration of nucleic acid.
Example 3 amplification of suicide gene HSV1-tk high fidelity KOD-Plus-Neo DNA polymerase was selected, HSV1-tk gene was PCR amplified from pORF-HSV1tk, the required NotI and SfiI restriction enzyme sites were introduced, the upstream and downstream primers were 5'-GCAGGCCCGATCGGCCATGGCCTCGTACCCCGGC-3' (the introduced SfiI restriction site underlined) and 5'-GCATCAGCGCGGCCGCTCAGTTAGCCTCCCCCAT-3' (the introduced NotI restriction site underlined), respectively, and the reaction conditions were 94 ℃ for 5min pre-denaturation; the following cycle was performed 30 times: 10s at 98 ℃, 30s at 62 ℃ and 1min at 68 ℃; then final extension is carried out at 72 ℃ for 5 min; and finally, storing at 4 ℃, amplifying the fragment at 1130bps, and separating by electrophoresis in 1% agarose gel after PCR amplification is finished, wherein the conditions are as follows: the voltage is 120V, and the time is 25 minutes; confirming the size of the band under an ultraviolet lamp as shown in figure 3, cutting a target band by using a clean blade, recovering and purifying target fragment DNA by glue, detecting the concentration of the recovered DNA by using a Nanodrop 2000, connecting a Blunt vector, carrying out competent transformation and connection of Trapn 1, coating the product on an ampicillin resistant plate, culturing the product in a constant temperature incubator at 37 ℃ for 12-14 hours, picking out a single clone in an ultraclean bench, carrying out double enzyme digestion verification on the plasmid by SfiI and NotI after small extraction, carrying out amplification culture on the clone with correct sequencing, extracting the plasmid, carrying out double enzyme digestion by SfiI and NotI to obtain an HSV1-tk gene fragment, recovering and purifying the glue, measuring the concentration, carrying out double enzyme digestion and purification on HS21/45vav1 by SfiI and NotI (figure 2), carrying out T4 connection on the purified HSV1-tk after concentration measurement, carrying out connection at 25 ℃ for 1 hour, transforming TransT1 cells, picking out single clone after culture, carrying out double enzyme digestion verification by SfiI and NotI to obtain a positive clone, the sequencing result is correct, and then the transgenic plasmid can be obtained: HS21/45vav1-HSV 1-tk.
Example 4 transgenic plasmids obtained above: HS21/45vav1-HSV1-tk is purified and then linearized, the plasmid is subjected to enzyme digestion by HindIII, the nuclear expression vector part in the vector is removed, the plasmid subjected to enzyme digestion is separated by 80V voltage, the gel is cut, QIAGENE DNA purified Kit is used for recovering a target fragment, and the purified linearized transgenic vector HS21/45vav1-HSV1-tk for microinjection is obtained.
Example 5 establishment and phenotypic characterization of transgenic HS21/45vav1-HSV1-tk mouse model: after obtaining the purified linearized transgenic vector HS21/45vav1-HSV1-tk for microinjection, integrating the linearized transgenic vector into a B6/C57 mouse cell genome by using a microinjection method, carefully breeding a pregnant mother mouse, producing a litter in 19-21 days, cutting toes and numbering after 3 weeks of the baby mouse, 500ul of tissue lysate, 8ul of protease K, carrying out overnight enzyme digestion at 55 ℃, adding 200ul of 5M NaCl after vortex mixing, adding 200ul of trichloromethane after mixing, centrifuging at 12000rpm for 5 minutes after mixing, carefully sucking the supernatant into a clean 1.5EP tube, adding 2 times of 95% ethanol in volume into the supernatant tube, mixing to see that the white floccule is genome DNA, centrifuging at 12000rpm for 5 minutes, discarding the supernatant, washing and precipitating twice at 1ml of 75% ethanol, centrifuging at 12000rpm for 5 minutes, carefully abandoning the supernatant, air-drying at room temperature for 10-20 minutes, dissolving the precipitate with 50ul of ddH2O to complete extraction of mouse genomic DNA, and designing HSV1-tk specific primers by using the extracted individual mouse genomic DNA: HS2,1-TK-F: GAAGCCGTGAGGGCGTAACT, TK-HS2, 1-R: TGTTCGCGATTGTCTCGGAA the integration of the target gene HSV1-tk is detected by PCR technology, as shown in figure 5, the target band is 750bps, and the first-built mouse for transducing HS21/45vav1-HSV1-tk is obtained.
Example 6 validation of the in vitro killing system: the proliferation condition CCK8 is developed after GCV gradient treatment; taking one female wild type B6/C57 and one female wild type Vav1-HSV1-tk-TG respectively, taking the spleen of the female wild type B6/C57 and one female wild type Vav1-HSV1-tk-TG under the aseptic condition, grinding the spleen to form single cells, filtering the single cells, adding a proper amount of erythrocyte lysate to perform lysis for 10 minutes at room temperature, adding anti-CD3 into a 1640 culture medium: 4ug/ml, IL-2:20ng/ml, anti-CD28:2ug/ml, resuspension, 20000 cells/well, inoculation into 96-well plate, culture at 37 ℃, 5% CO2, GCV concentration gradient treatment at day 3, results show: as shown in FIG. 6, when spleen cells of model mice were cultured in vitro, the color of CCK8 after GCV treatment gradually faded with the increase of GCV treatment concentration, which indicates that the number of living cells gradually decreased with the increase of GCV treatment concentration, and is consistent with the statistical results of OD630nm-OD450 nm.
Example 7 detection of CD3+ cell survival after GCV treatment with concentration gradient, collecting GCV treated cells of different concentrations into 15ml centrifuge tube, 400g centrifugation for 5 minutes, PBS washing once, transferring to clean 1.5EP tube, 50ul staining buffer resuspending cells, 1ul Fc sealing, room temperature 10 minutes, adding the antibody CD45, 1ul, CD3, 5ul, mixing well, 4 ℃ photophobic staining for 30 minutes, simultaneously setting up single color staining CD45, CD34 cell tube, shaking once every 15 minutes, after staining, 1ml PBS washing, 400g centrifugation for 5 minutes, during the preparation of dead and live staining solution, in this scheme using Annexin V-PI double staining method to detect cell survival rate, 4Xb buffer using ddH2O to dilute to 1X, each reaction 300ul volume preparation solution, adding PI 10 ul/reaction, Annexin 5ul reaction dye reaction, and (3) dyeing for 15 minutes at room temperature in a dark place, simultaneously setting a monochrome dyeing PI cell tube, an Annexin V cell tube and a blank cell tube which is not dyed, performing on-machine detection in a flow manner within 1 hour, and obtaining a result from a statistical graph 7 of the detection result: with the increase of the GCV treatment concentration, the survival rate of CD3+ cells is gradually reduced, which shows that the killing effect of the HSV1-tk/GCV system is effective, but the survival rate of CD3+ cells of a WT mouse is obviously reduced due to 80uM, which shows that the 80uM concentration has stronger cytotoxic effect.
Example 8GCV concentration gradient post-treatment flow assay, apoptosis-related protein assay statistics, harvesting GCV-treated mononuclear cells of different concentrations, placing on ice, 100ul of RIPA plus PMSF lysing the harvested cells, 30 min lysing on ice, 12000rpm, centrifuging at 4 ℃ for 15 min, carefully harvesting supernatant into a clean 1.5EP tube, labeling, protein quantification by BCA method, calculating sample concentration, sampling at 20ug, diluting and adding 5XLoding, replenishing RIPA, finally adding 20ug of protein sample, centrifuging to the bottom of the tube, boiling at 100 ℃ for 10 min, placing the boiled sample at-20 ℃ for future use; preparing 12% SDS-PAGE gel, carefully dropping protein samples, recording the sample adding sequence, carrying out 80V electrophoresis on concentrated gel for 30 minutes, carrying out 120V electrophoresis on separation gel for 1-1.5 hours, carrying out wet transfer on the gel, carrying out 230mA transfer on the gel for 70 minutes, dissolving 5% skimmed milk in TBST, sealing at room temperature for 1 hour, washing the TBST once, incubating at 4 ℃ overnight once, washing the membrane for 3 times at TBST on the next day, carrying out 10 minutes each time, incubating at room temperature for 1 hour for secondary antibody, washing the membrane for 3 times at TBST, and carrying out ECL luminescence and color development 10 minutes each time. From Western blot and grayscale analysis results fig. 8, it can be seen that: after the Vav1-HSV1-tk mouse mononuclear cells are subjected to GCV gradient treatment in vitro, the protein level of activated caspase 3 is increased and is GCV concentration-dependent, further explaining the effectiveness of the killing system, and consistent with the flow analysis result of 4.2, the 80uM treatment group does not show linear increase, which indicates that the toxicity to cells is overlarge at the concentration of GCV.
Example 9 validation of killing system in vivo of positive transgenic mouse F1, dividing the F1 generation mouse into two groups of 3 mice each, injecting GCV80mg/kg intraperitoneally, administering 200 ul/one/day ddH2O to the control group, analyzing the ratio change of peripheral blood lineages for one week, and the experimental design is shown in FIG. 9.
Example 10 validation of the in vivo killing system of mouse F1, PCR of the peripheral blood genome identified the presence of HSV1-tk gene one week after GCV administration. PCR detection of HSV1-tk agarose gel results FIG. 10 shows: in the GCV-treated group, the presence of HSV1-tk gene was not detected, and the transgenic mice were able to effectively clear the autologous blood system after GCV treatment.
Example 11GCV maximum safe dose exploration experiment, 50 normal wild type B6/C57 male and female mice were randomized into 5 male groups and 5 female groups of 10 mice each, and the doses were: PBS, 20mg/kg, 40mg/kg, 80mg/kg, 160mg/kg, intraperitoneal injection was performed every day, and the body weight of each group of mice was measured every three days, wherein the body weight monitoring results of each group of mice are statistically known in FIG. 11: with the increase of GCV administration dosage, the weight of the mice is obviously reduced when the GCV treatment dosage is 160mg/kg, which indicates that the dosage generates obvious toxic and side effects on the mice.
Example 12 two weeks after treatment in the GCV maximum safe dose discovery experiment, mice were sacrificed and the organs were carefully removed, weighed, fixed and the weight statistics are shown in fig. 12: consistent with the statistical results of body weight, the weight of each organ of the mice is obviously reduced when the GCV is used at a large dose of 160mg/kg, which indicates that the mice have obvious toxic reaction under the GCV treatment condition at the dose.
Example 13 fig. 13 is a graph of a representative flow analysis of a flow analysis of the correlation between peripheral blood and bone marrow leukocyte viability in a GCV maximum safe dose discovery experiment, from top to bottom: the first column is a single-stained sample of CD45, the second column is an isotype control stained group of CD45 antibody, the third column is a positive control group of apoptosis staining after H2O2 treatment, and the last column is a representative whole-stained sample group.
Example 14 is a statistical graph of flow analysis results relating to the survival rate of leukocytes in peripheral blood and bone marrow by flow analysis every three days in the GCV maximum safe dose exploration experiment, the flow analysis strategy is shown in FIG. 13, the abscissa is the time point, the ordinate is the survival rate of leukocytes of mice in the experimental group relative to the control group, and it can be seen from the statistics of blood cell survival rate in FIG. 14 that there is no significant difference in the survival rate of blood system cells of mice in the concentration range of 0-160mg/kg of GCV.
Example 15 figure 15 is a flow analysis strategy diagram after chimeric replacement transplantation in an experiment for obtaining allogeneic blood system chimeras, positive F1 generations of HS21/45vav1-HSV1-tk-C57(CD45.2) mice were transplanted whole bone marrow into myeloablative irradiated wild-type normal B6/C57(CD45.1/2), after 4 weeks, flow analysis of the distribution of individual lineage cells of blood in peripheral blood revealed that HS21/45vav1-HSV1-tk positive cells (CD45.2) had successfully replaced naive cells (CD45.1/2), chimeric replacement was performed with wild-type normal B6/C57(CD45.1) mouse bone marrow cells 2 weeks after GCV treatment of mice for 2 weeks, during which GCV treatment was continued for 4 weeks, and at the same time, serial transplantation was performed once a week apart, after GCV treatment for 4 weeks, mice were sacrificed 4 weeks, and peripheral blood flow-tested groups of mice were sacrificed, and the proportion of chimeric replacement cells in bone marrow, wherein the PBS treatment group is a control group, the GCV is an experimental group, peripheral blood is detected once a week in a flow mode, and the long-term observation results are integrated, so that the GCV can well eliminate recipient bone marrow cells after the chimeric replacement transplantation and after the chimeric replacement transplantation, the GCV is treated for 4 weeks respectively, and allogeneic hematopoietic chimerism is realized. The flow analysis mainly analyzes the proportion of CD45.1 and CD45.2 positive cells and the proportion of T cells, B cells and myeloid cells in CD45.1 and CD 45.2.
Example 16 statistical analysis of the variation in absolute number of individual lineages in CD45.1, CD45.2 after 7 chimeric replacement transplants in experiments for allogeneic blood system chimera, 80-M-110, 80-M-111, 40-M-113 are representative of three experimental groups of mice, with cell types of individual lineages on the ordinate and absolute cell numbers on the abscissa. From the statistical results fig. 16, it can be seen that: the statistics of 7 consecutive days show that the positive proportion of CD45.1 in each lineage cell in peripheral blood shows a gradually rising trend as a whole with the prolonging of the GCV treatment time.
Example 17 representative flow analysis plots for the proportion of CD45.1 positive cells in hematopoietic stem cells in bone marrow cells were examined after 4 weeks of cessation of GCV treatment following chimeric replacement transplantation in experiments to obtain allogeneic blood system chimeras. The change in the proportion of CD 45.1-positive cells in the Lin-sca1+ c-kit + cell population among the live cells was analyzed primarily in this assay.
Example 18 acquisition of allogeneic blood system chimeras after 4 weeks of GCV treatment stopped following chimeric replacement transplantation in the experiment, mice were sacrificed, the femurs of the mice were harvested, whole bone marrow was washed out with PBS, FC was blocked for 10 minutes at room temperature, antibody stained, protected from light for 25 minutes, washed once with PBS, tested in an on-machine flow format, and the proportion of CD45.1 positive cells in the Lin-sca1+ c-kit + (LSK) cell population in bone marrow cells was determined. According to the statistical result, the following results are obtained: the LSK + CD45.1+ ratio of three representative chimeric mice is predominant, and the CD45.1+ cell ratio reaches nearly 80% in the overall level.
Although the invention has been described in detail with respect to the general description and the specific embodiments, it will be apparent to those skilled in the art that modifications and improvements can be made to the invention or the method can be practiced without the specific embodiments. Accordingly, it is intended that all such modifications, improvements and extensions that do not depart from the spirit of the invention, be considered within the scope of the invention as claimed.
Sequence listing
<110> institute of animal research of Chinese academy of sciences
Preparation method and application of <120> HSV1-tk/GCV induced blood system defect mouse model
<130> 1
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2466
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
gaaattcgaa tctagaaacc tagtgggcgc tctccagctt gcctctcgtt tttgttttgt 60
tttcactgcc tggccttcct accctaactg cgtgcaaaga aaacaaattg ttacccggtt 120
tagagaagag ggagaggacg cgtaggaagt cacagacgcc aaaaataggg gatagaaatt 180
gtcctcgcct gtaaggttgc aaaacgggag ggttttcagg ttgtgcccac ccctacccat 240
atcctcagtg cgtaaatgag actcaagctg gtccagcgaa gccgtttagc gccgcctaga 300
ggccataagg ataaaaggtg gggtgctaag gaagcggctt ggaatctggt gggttagtca 360
gggagaggct tgcttgccaa ggtaatttta gttcctgaga agctatggtc aggaaagcag 420
tacatatttg ttagacaaat caactaataa atgacaggga agggcactgg tgaccttgac 480
tcggacgaga actcacagca gggaaggtgg gcttatagta tgataattaa gggtggagga 540
gggctgtggc ttaaaattta ggaagactta agggaaaaaa aatgacattt gggggctggg 600
gagatggttc agtcaaatgc ttgctactca aatgagagga caagagttca aatccctagg 660
gcacaattaa agcaggagga ggtagtctgt gctctttaat ctcagtgctc ctaagacgag 720
atgggggcag agacaattct ggaagttcag ctagccagga gcagctagcc caggagtaag 780
agagactcta tgttaagtta agtgtgtgga agagagggtc tacactgagg ctgtcctgcc 840
acctcaacac acactccata gtgttctctc tctctctctc tctctctctc cttctttctc 900
tctctctctc tctctctctc tctctctctc tctctctctc tctttctcac acacacacat 960
acaagtttta aagaaaaaag gaaaagtggg tggtgagggt gagagggctc tgtgggcaaa 1020
ggtgattgct accaagccta atgatctgag ctcaatcgtt ggggcttgca tggtggaaag 1080
aggacctact cccttgggct ggtgagatgg ctcagtgggt tagagcacct gactgctctt 1140
ccgaaggtcc agagttcaaa tcccagcaac cacatggtgg ctcacaacca tccgtaacaa 1200
gatctgaccc cctcttctga agtgtctgaa gacagctaca atgtacttac atataataaa 1260
taaataaata aataaatctt taaaaaaaaa aaaaagagga cctactcccc catgttaacc 1320
tttgacctcc acacatgtac tgtgccccac atgtaccttc acacatacac acatacatat 1380
gtatatatat acacacacac acatatatat atgtaataaa agtgaaatgg ctttcaaagt 1440
agaatatatt ttctaatgtt tattatagtt ctatttgttg gcagtttcct acatgttaac 1500
aacctatctt cgtcctaccc accctcaact cccctctgac tccgaatgag agtggactct 1560
ctttacctta tccccacttt cttgctcttt tgtaaccctt tgcatccatt actgctgcct 1620
gtgggcatgt tggctccctc ctggcttcgt cttgtgctgt caccataact gcacggagtt 1680
catgagcgta gtggccatgg gtatgccctg gagatagcat ttcacatccc ccgcccccga 1740
ctcttaccga ctttctgttc cttctccctt gagcctcggg ccggtggtgg tatagatgtc 1800
ccatctatgg ctgagcactc agtaatcaaa aagtaacatg taagaagtga tggagtgaat 1860
tagtgtcttc tgtcaggtgg tgggcacaag tgcaaaggcc ctggggcagg atggaggtca 1920
gggagtgaag aacagagaga ggagaagagg ctggcagggg ttggggcagg gtgaacaggg 1980
ccttgtgggg tgcaggaaag acatgagttt tggccccaaa gcaaggttga accaggaggg 2040
ttatgggtgg tggaagaatg gttgagactt gactcagtgt tcagtggtcc cttttggctg 2100
ctgcaggggc aaaaggctgt gagagatcag ggccgtagac tggggaccag gaggagacag 2160
gaggtgacag cctaggtgcc tacggctgga tgagatagtg gcaccttagt tcagcaggtg 2220
cccaggtctg ctcagtgtag gtggagaaag acgaggtcac accagtgagt ggaagccgtg 2280
agggcgtaac tgcagatgct gctccccctc cgccccacag ctcctcccca cccccacccc 2340
ggggcgacag ttacagtcac agaagaggaa gtggtgttgt agttgtcccc actggggcag 2400
gcggcggcgg tggtgaagga acgagggtgc acggggcctc cccgaggggc caagtgagag 2460
gccggc 2466
<210> 2
<211> 1131
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
atggcctcgt accccggcca tcaacacgcg tctgcgttcg accaggctgc gcgttctcgc 60
ggccatagca accgacgtac ggcgttgcgc cctcgccggc agcaagaagc cacggaagtc 120
cgcccggagc agaaaatgcc cacgctactg cgggtttata tagacggtcc ccacgggatg 180
gggaaaacca ccaccacgca actgctggtg gccctgggtt cgcgcgacga tatcgtctac 240
gtacccgagc cgatgactta ctggcgggtg ctgggggctt ccgagacaat cgcgaacatc 300
tacaccacac aacaccgcct cgaccagggt gagatatcgg ccggggacgc ggcggtggta 360
atgacaagcg cccagataac aatgggcatg ccttatgccg tgaccgacgc cgttctggct 420
cctcatatcg ggggggaggc tgggagctca catgccccgc ccccggccct caccctcatc 480
ttcgaccgcc atcccatcgc cgccctcctg tgctacccgg ccgcgcggta ccttatgggc 540
agcatgaccc cccaggccgt gctggcgttc gtggccctca tcccgccgac cttgcccggc 600
accaacatcg tgcttggggc ccttccggag gacagacaca tcgaccgcct ggccaaacgc 660
cagcgccccg gcgagcggct ggacctggct atgctggctg cgattcgccg cgtttacggg 720
ctacttgcca atacggtgcg gtatctgcag tgcggcgggt cgtggcggga ggactgggga 780
cagctttcgg ggacggccgt gccgccccag ggtgccgagc cccagagcaa cgcgggccca 840
cgaccccata tcggggacac gttatttacc ctgtttcggg cccccgagtt gctggccccc 900
aacggcgacc tgtataacgt gtttgcctgg gccttggacg tcttggccaa acgcctccgt 960
tccatgcacg tctttatcct ggattacgac caatcgcccg ccggctgccg ggacgccctg 1020
ctgcaactta cctccgggat ggtccagacc cacgtcacca cccccggctc cataccgacg 1080
atatgcgacc tggcgcgcac gtttgcccgg gagatggggg aggctaactg a 1131
<210> 3
<211> 796
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gttaacttgc ggccccagat gtcccaggtg agtctgctgg gtgatgctag ggcccgactg 60
gataaaggct ggtaggcccg gctctggcta tggagaggtg tcgagcagaa cggagctctg 120
ggtgggtttg tacactcttg gttggcttgc agctgtccca gcaaatttag ggggaaatct 180
ccctatcccg ccccgccccc agctgactct gttgagatcc atttaaaggg gtcctaagag 240
gtatcttgtt gcctcgtagt tagccaagag ggtgcatgag gttggagaac cagattcaag 300
agagagaggc cttcagtttc tcccccaccg gtggctcctc ctctaccacc tcttctgccc 360
ccctcctcct cacatcttcc ttcacctcca tttcctgcca gggctttgtg ctgtgtggcg 420
tggagtgttg ggggctgcaa tgcgagaccc tgtggcatcc caactctacc tatttcggga 480
agggcccatt gaagtgcagg gcccaggcct agggtcctaa caaggccctg ggtgtccctg 540
agtttgacat ctgtgcactc tccgtgtttc aagtggaggc atggtcagac tatgccccgg 600
tgagcctaga aaatgtattc aaatccttat ttgaatgtgg ctggggtggt ggtgaaaggt 660
gactggtggc tttgtactct tttctctgct tccccaggga tgctgggttt cggggggagg 720
gctgatagcc ctagaaatga aaggtatagg ttcaagtcaa agggatcgag atctagatat 780
cgatgaattc gagctc 796
<210> 4
<211> 3859
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggtacctgcc gtcatgtatc cacacacagc ccttcactgg tggattctag gcagggctcc 60
acctctgacc tcgtccccag tccctcacta gtgaatctag ggtaggggct ccactcagag 120
tcacatccca gcccttagag atttttatat tttgagtgga gctgattgtc aggagtctag 180
gttgcacagc tttgtgtgtg tcactgtaga tgttcggaga ctcttggttg ttttttgttt 240
tttcaagaca gggtttctct gtgtagccct ggctgtcctg gaactcactc tgtagaccag 300
gctggccttg aacccagaaa tctgcctgcc tctgcctccc aagtgctggg attaaaggcg 360
tgcgccatca cgcccagctt agagactctt ttttttaaag tggaagcacc tgtaccttgg 420
aaggtcaccc cgtccagcct tctcccatac actgtccatt accgctctga cttgctgtgg 480
ctgccagctg agccccactt tcccctgcac taccccacag ctgtccagtt gtctgtcctt 540
gttactgtgt acctcagctg tctgtacttc ctatcacaca ctccagttgc tgttaccaat 600
tgatacccag attgctttgt gctggaagcc cctcctgttt atctacccca cctctccagg 660
ttataagcac tggggcatca gggtcatgtt ttcctcaccc actactgggt ctcagggctg 720
ggtaaacagt aaatgcttaa tatatgcttt tggagtcaac taatgtctgg gtttttggtc 780
aggctttgtg ttagtgctca gttctaaagt ggagaggttg attctccttc tctctgttct 840
tctggctttc gccttctctc cctctctcct tctttccttt tctcctcttt ctctgttctt 900
cttccaaccc ctccacattt gtccctctcc ttcccttcct attccctcat cttttatcgt 960
atgaataaca ttttgtctcc tgggggagat gtgaacacgt gtctactcgt ctcagacagg 1020
gccaacagcc aggaaaagca aggatcccgc caaagtccaa cttgatgaag cactgagtgt 1080
cactgtgtta cagataggaa tgtgggtcag ggctcactta acaggaacag aaatggctca 1140
aatacagctg tatcaccaaa gcctggcatg gatggtgggc gactcaggaa agctgccttc 1200
aggcagctct gaaaggtgga cagttgtctc ttctaggtgc cttgtcctgt ctgaacctct 1260
tctgggcagt tcagcttgtc ttgagagcgg tcaccagtag tccttacagc ttgtgcttgg 1320
ggagggcagt gcctagagaa tctggtctgt ttcaggaact tcctgcatat ctcctgttgt 1380
ttcctgagct tcacgggctt ttctgtaggc tagaatgctt cacctccact tagcacatcc 1440
tatgtcttaa tgagcttcct tccaaggtgg aaggtttcgt cttaagggaa attgctacac 1500
agcaccctct ttccccctcc ctctgccttc tctcccttcc actatcttct cccctcaccc 1560
ctgtttccca cccctgtttt tgacacaggg tctcactgtg gttccctgga caacctggaa 1620
ctcactctgt agtccatgct taccccagag tttagccatc ctttgctctg cctcctcttc 1680
ctgagcgcgg agattacagg tatattccac cacactggga tttcatttct gatttttggt 1740
tggagaaccc aaggcaggat gacctgtcca aggtccccct gcttaccagt ttcagctggg 1800
agtcaccaga cacctagttg tggaaggcgt actggggtgt gaggtgacag ggaaagaaga 1860
ggaagtctga ttcccagaat gctttagact catgctgaag agttgggtca tcagctcctc 1920
aattcaaagc tctggacagg gtgagcagcg tgaccacccg ctctgggaag cctcagggag 1980
ctagctggag tctgaaagat tacagggctg aaggaaggca gagagccatg cctctacccc 2040
ttgtctatct tccccagcca ggcctggtaa caggcatttc ctgtttccgg ccatctcctg 2100
ggtttgggtt gtgggtgtgg aaagcgcaga aggcagaatt tgtgttgagt ctgggccgct 2160
ttctcccagc ccccaagaga ggccactgtc cttactgcta cttccttgaa cagaattctc 2220
tctattttgg gcagaggcac caaaagccaa cttccggctt gatcacagtg cctggctcct 2280
gatttgccta aagccttctt gcttgtctag cgccatggtc ttctttcgtc ccggaggata 2340
agggaagcag aggggttgtc cctataaaac aaggaagaat tctccttcca cgtgcctcct 2400
acagtgagtg gcttcctgtc cttcatcatc ttttcacatg ccttacggtg ccaggaagtt 2460
cttctggtgg tctaaccaca gtcaccgtgc tttctgaagg agcccagctc tctaactagc 2520
tactctgaag cggtggaagg ggttatgaat catactcctt ttggaagagt catgtgactt 2580
gagggaaggc agtctcaaag ctcagctgat tctctgaggt taggaggggg atttccttcc 2640
ttaggccaca cagacctaag gccagacatc acctctggtg ttggtctgcc agccctgtct 2700
gcctgtctat ggcccacagg acttctgagt gctgtatata atggtgtata taatttctgt 2760
gatatgctgg tccctgttat gtccccgtga ggcctgggcg agggggctgt ggtggctcaa 2820
tgtggttcag gagctagggt agagcaggag tggggctggg ttctgtgtgt ttctccacac 2880
tcctgctttt acatgtgtga ctctggcagg tgacagccct gtgacaagcc tcagttttcc 2940
cacctgtcag atagaagtcc tttgtttagc catttcgtct ttcacttgtg cgtgtgtgta 3000
tttgtatgca tttgtgcatg tttattatta ctattatatt gtgtgtgtgt gtgtgtgtgt 3060
gtgtatgggt gcagatgcct gaggaggcca gaagagggca ttggatcctc ctgagttgga 3120
ctcacaagtg tgtgtgatat gggatggaaa ccaaacttga ctcctctgtg agatggacag 3180
tgccagcacc cctgaacatt ctcgtgggtt taagcattga acccagggcc ttgctttagc 3240
aattgagttg cctctcagac ctccttcaat aacttagaaa ttagagcata tttttttttt 3300
ggcagtttgg tctgtgtaca tatgtatttt gatcatggcc gctctactcc cttgtctttt 3360
ttgtgttgtt gttgttgttg tttttttttt tttgtgtgac ccagtgagtt gaattagagt 3420
ttgtgacagg agtcagggtg aggggtcatg taccagtggc tgcaacactg aagaaaatgc 3480
ctctcccttc cctggaagct gttagctgcc tacaaatccc cagggaggag tgggtcctca 3540
tttgactccc cactccttca acacaacaac ttctcttgag ctccacccca gccccaggtc 3600
tttaaatagt tgtccacatg gaagagcgag cccttgagtg acctgtggcc ttaggtaatg 3660
gtggagtgtc atgtagggtt gatgactgtc acaagtggac cagttgtctg tggggagttg 3720
atggtgctga ggctgtatac atggagacag agctccattt ggctgacaat ccaagatagc 3780
tgaaaaaaaa tagtttaaat ctggggctgt gcctcaatgg gaagaacatg tgtccagtgt 3840
gcacagagtc ctgggtacc 3859
Claims (10)
1. The recombinant vector expression unit is characterized by comprising a Vav1 promoter, three structural domains of HS2/1, HS4 and HS5 and a suicide gene HSV 1-tk.
2. The recombinant vector expression unit of claim 1, wherein the recombinant vector expression unit comprises a Vav1 promoter-HS 2/1, a suicide gene HSV1-tk, a Vav1 promoter-HS 4 and a Vav1 promoter-HS 5, which are connected in sequence.
3. The recombinant vector expression unit according to claim 1 or 2, wherein the recombinant vector expression unit is constructed as follows: HS2/1, HS4 and HS5 structural domains of Vav1 gene and suicide gene HSV1-tk are amplified, and the suicide gene HSV1-tk is inserted between Vav1 promoter-HS 2/1 and Vav1 promoter-HS 4 according to the sequence of Vav1 promoter-HS 2/1, Vav1 promoter-HS 4 and Vav1 promoter-HS 5.
4. The recombinant vector expression unit according to any one of claims 1 to 3, wherein the nucleotide sequences of the Vav1 promoter-HS 2/1, suicide gene HSV1-tk, Vav1 promoter-HS 4 and Vav1 promoter-HS 5 are shown in SEQ ID nos. 1 to 4, respectively.
5. A vector comprising the expression unit of any one of the recombinant vectors of claims 1-4.
6. A construction method of a transgenic animal model is characterized by comprising the following steps: integrating the vector of claim 5 into the genome of an animal.
7. The construction method according to claim 5, characterized in that the construction method specifically comprises: extracting and purifying the vector of claim 5, carrying out linearization treatment, integrating a target gene into an animal genome by utilizing fertilized egg prokaryotic injection, transplanting an oviduct embryo into a surrogate pregnant mouse, obtaining a first-built mouse after 21 days, cutting off 2 mm of mouse tail when a newborn mouse grows to two weeks, extracting the mouse tail genome by an alkaline lysis method, designing HSV1-tk specific primers, carrying out genotype identification, and obtaining the positive first-built mouse with an HSV1-tk target band.
8. A method for producing a chimeric animal model, which comprises the step of transplanting whole bone marrow of the positive naive mouse obtained in claim 7 to a wild type normal mouse which has been subjected to an irradiation treatment.
9. Use of an animal model prepared according to any one of claims 6 to 8 in xenogeneic organ transplantation or xenogeneic blood system fusion or recipient bone marrow depletion or xenogeneic organ reconstruction or immune tolerance mechanism studies.
10. Use of an animal model prepared according to any one of claims 6 to 8 as a model animal for the study of the cellular function and phenotype of the hematopoietic and immune systems.
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Citations (3)
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US20100233144A1 (en) * | 2007-10-12 | 2010-09-16 | Universite Pierre Et Marie Curie (Paris 6) | Method for optimizing blood cell transplants |
CN101899445A (en) * | 2010-06-23 | 2010-12-01 | 中国医学科学院血液病医院(血液学研究所) | Specificity overexpression mouse model of transduction iASPPsv cancer gene in hemopoietic system and preparation method and use thereof |
US20150299278A1 (en) * | 2012-01-13 | 2015-10-22 | Sloan Kettering Institute For Cancer Research | Cancer-specific suicide gene for cell-based and gene therapy |
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US20100233144A1 (en) * | 2007-10-12 | 2010-09-16 | Universite Pierre Et Marie Curie (Paris 6) | Method for optimizing blood cell transplants |
CN101899445A (en) * | 2010-06-23 | 2010-12-01 | 中国医学科学院血液病医院(血液学研究所) | Specificity overexpression mouse model of transduction iASPPsv cancer gene in hemopoietic system and preparation method and use thereof |
US20150299278A1 (en) * | 2012-01-13 | 2015-10-22 | Sloan Kettering Institute For Cancer Research | Cancer-specific suicide gene for cell-based and gene therapy |
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A. SANCHEZ-AGUILERA等: "Guanine nucleotide exchange factor Vav1 regulates perivascular homing and bone marrow retention of hematopoietic stem and progenitor cells", 《PROC NATL ACAD SCI U S A》 * |
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