Method for preparing beta<IVS-II-654> transgene mouse model carrying human beta globin gene

Abstract

The invention discloses a method for preparing a beta<IVS-II-654> thalassemia transgenic mouse model carrying human beta-globin gene. A lentiviral vector mediated human beta-globin gene is stably integrated in the genome of the mouse model, and the genome can be expressed continuously and stably; furthermore, the genome can also be inherited to the offspring and maintain the original expression level, thus phenotypes such as hematology, pathology and the like of the beta<IVS-II-654> thalassemia transgenic mouse model carrying the human beta-globin gene are improved. The mouse model prepared by the method can be used for evaluating the effectiveness, the stability and the security of the lentiviral vector mediated human beta-globin gene in the treatment of beta<IVS-II-654> thalassemia.

Description

technical field

[0001] The invention belongs to the field of biotechnology, in particular to a beta globin gene carrying human beta globin IVS-II-654 Preparation method of transgenic mouse model. Background technique

[0002] Beta thalassemia is one of the most common monogenic disorders, with approximately 80 million carriers worldwide. β-thalassemia is mainly a chronic anemia caused by a point mutation or deletion of the β-globin gene, resulting in reduced or complete lack of β-globin chain synthesis. It is widely prevalent in the Mediterranean region, the Middle East, Southeast Asia and Africa, and the southern provinces of my country are also high-incidence areas . Severe beta thalassemia manifests as impaired erythropoiesis and severe anemia requiring lifelong blood transfusions to sustain life. However, due to the deposition of iron ions, the patient eventually died of organ failure.

[0003] beta IVS-II-654 Thalassemia is a mutation type of β-thalassemia unique to...

Examples

Embodiment 1

[0026] Example 1 , Construction of human β globin gene lentiviral expression vector and viral packaging

[0027] like figure 1 As shown, construct human β globin gene lentiviral expression vector, specifically as follows

[0028] 1.1 Cloning of human β-globin gene

[0029] According to the genome sequence of human β-globin (β-globin) published by Genebank, the primer pair HP1 and HP2 were designed, and its sequence is as follows:

[0030] Primer HP1: 5'-AGGTTGCAGTGAGCTGAGAT-3';

[0031] Primer HP2: 5'-GCGAGCTTAGTGATACTTGT-3'.

[0032] Using normal human genomic DNA as a template, amplify the human β-globin gene fragment (including 3 exons and 2 introns), as follows:

[0033] PCR reaction system: 2.5μl 10×PCR buffer, 2μl MgCl 2 (25mM), 2μl dNTP (2.5mM), 0.6μl primer (10pmol / μl), 0.2μl Taq enzyme (5U / μl), add double distilled water to 25μl.

[0034] PCR reaction conditions: denaturation at 94°C for 5min; (94°C for 45sec, 60°C for 45sec, 72°C for 60sec) x 32 cycles; 72°C ...

Embodiment 2

[0047] Example 2 , Preparation of transgenic mice

[0048] The LBG virus particles obtained in Example 1 were injected into wild-type mice and β 654 The perivitelline space (under the zona pellucida) of fertilized eggs obtained from mating mice, each fertilized egg was injected with 5×10 -4 μl of virus suspension, and then the fertilized eggs were transplanted into the oviducts of pseudopregnant mice, developed in utero to birth, and two different types of primary transgenic mouse models were obtained. The primary mice F0 were mated with wild-type mice to generate offspring mice F1 and F2.

Embodiment 3

[0049] Example 3 , Identification of transgenic mice

[0050] After the mice were born, the tails of the mice were clipped to extract genomic DNA, and then PCR reactions were performed to design LTR (detection of lentivirus), β-major (detection of mouse β-major gene) and β654 primers to detect the genotype of mice.

[0051] The PCR primers are as follows:

[0052] LTR-1: 5'-GACTTACAAGGCAGCTGTAG-3';

[0053] LTR-2: 5'-GTACAGTCCGGATGCAGCTC-3'.

[0054] Product length: 586bp

[0055] β-major-1: 5′-AGGCAGCTCACAAGAAGAAG-3′;

[0056] β-major-2: 5'-TGGAGACTGCTCCCTAGAAT-3'.

[0057] Product length: 421bp

[0058] β654-1: 5'-AGTGATAATTTCTGGGTTAAGGT-3';

[0059] β654-2: 5'-AGGGCCTAGCTTGGACTCAG-3'.

[0060] Product length: 179bp

[0061] PCR reaction conditions: denaturation at 94°C for 5min; (94°C for 45sec, 60°C for 45sec, 72°C for 60sec) x 32 cycles; 72°C for 10min.

[0062] The PCR product was detected by 2% agarose gel electrophoresis, the results are shown in figure 2 ...

technical field

[0001] The invention belongs to the field of biotechnology, in particular to a beta globin gene carrying human beta globin IVS-II-654 Preparation method of transgenic mouse model. Background technique

[0002] Beta thalassemia is one of the most common monogenic disorders, with approximately 80 million carriers worldwide. β-thalassemia is mainly a chronic anemia caused by a point mutation or deletion of the β-globin gene, resulting in reduced or complete lack of β-globin chain synthesis. It is widely prevalent in the Mediterranean region, the Middle East, Southeast Asia and Africa, and the southern provinces of my country are also high-incidence areas . Severe beta thalassemia manifests as impaired erythropoiesis and severe anemia requiring lifelong blood transfusions to sustain life. However, due to the deposition of iron ions, the patient eventually died of organ failure.

[0003] beta IVS-II-654 Thalassemia is a mutation type of β-thalassemia unique to...

Examples

Embodiment 1

[0026] Example 1 , Construction of human β globin gene lentiviral expression vector and viral packaging

[0027] like figure 1 As shown, construct human β globin gene lentiviral expression vector, specifically as follows

[0028] 1.1 Cloning of human β-globin gene

[0029] According to the genome sequence of human β-globin (β-globin) published by Genebank, the primer pair HP1 and HP2 were designed, and its sequence is as follows:

[0030] Primer HP1: 5'-AGGTTGCAGTGAGCTGAGAT-3';

[0031] Primer HP2: 5'-GCGAGCTTAGTGATACTTGT-3'.

[0032] Using normal human genomic DNA as a template, amplify the human β-globin gene fragment (including 3 exons and 2 introns), as follows:

[0033] PCR reaction system: 2.5μl 10×PCR buffer, 2μl MgCl 2 (25mM), 2μl dNTP (2.5mM), 0.6μl primer (10pmol / μl), 0.2μl Taq enzyme (5U / μl), add double distilled water to 25μl.

[0034] PCR reaction conditions: denaturation at 94°C for 5min; (94°C for 45sec, 60°C for 45sec, 72°C for 60sec) x 32 cycles; 72°C ...

Embodiment 2

[0047] Example 2 , Preparation of transgenic mice

[0048] The LBG virus particles obtained in Example 1 were injected into wild-type mice and β 654 The perivitelline space (under the zona pellucida) of fertilized eggs obtained from mating mice, each fertilized egg was injected with 5×10 -4 μl of virus suspension, and then the fertilized eggs were transplanted into the oviducts of pseudopregnant mice, developed in utero to birth, and two different types of primary transgenic mouse models were obtained. The primary mice F0 were mated with wild-type mice to generate offspring mice F1 and F2.

Embodiment 3

[0049] Example 3 , Identification of transgenic mice

[0050] After the mice were born, the tails of the mice were clipped to extract genomic DNA, and then PCR reactions were performed to design LTR (detection of lentivirus), β-major (detection of mouse β-major gene) and β654 primers to detect the genotype of mice.

[0051] The PCR primers are as follows:

[0052] LTR-1: 5'-GACTTACAAGGCAGCTGTAG-3';

[0053] LTR-2: 5'-GTACAGTCCGGATGCAGCTC-3'.

[0054] Product length: 586bp

[0055] β-major-1: 5′-AGGCAGCTCACAAGAAGAAG-3′;

[0056] β-major-2: 5'-TGGAGACTGCTCCCTAGAAT-3'.

[0057] Product length: 421bp

[0058] β654-1: 5'-AGTGATAATTTCTGGGTTAAGGT-3';

[0059] β654-2: 5'-AGGGCCTAGCTTGGACTCAG-3'.

[0060] Product length: 179bp

[0061] PCR reaction conditions: denaturation at 94°C for 5min; (94°C for 45sec, 60°C for 45sec, 72°C for 60sec) x 32 cycles; 72°C for 10min.

[0062] The PCR product was detected by 2% agarose gel electrophoresis, the results are shown in figure 2 ...

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