Preparation method and application of HSV1-tk/GCV induced blood system defect mouse model
A construction method, -HS4 technology, applied in the field of mouse model preparation, can solve the problems of unrealized reconstruction of the human immune system, high requirements for feeding conditions, short survival period, etc., and achieve the effect of removing immune barriers and physiological barriers
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Embodiment 1
[0049] Example 1 According to the specifically related structural domains of the Vav1 gene in the hematopoietic system, it was found that they were HS2 / 1, HS4, and HS5, and the target gene HSV1-tk was structurally designed with the Vav1 gene promoter region specifically expressed in the hematopoietic system. Such as figure 1 .
Embodiment 2
[0050] Example 2 The results of the PCR of each fragment of the backbone vector HS2 / 1, 4, 5 and vav1 and the linearization products of SfiI and NotI double digestion are as follows figure 2 The specific implementation method is: using high-fidelity KOD-Plus-Neo DNA polymerase, using the mouse genome as a template to perform PCR amplification to obtain various functional domains, T4 connection, TransT1 competent transformation, and plasmid extraction for positive clones after identification, SfiI and NotI 37°C double enzyme digestion linearization, 1% agarose gel electrophoresis separation at 120V voltage for 25 minutes, then DNA gel recovery, nucleic acid concentration was measured and stored at -20°C for later use.
Embodiment 3
[0051] Example 3 Amplification of suicide gene HSV1-tk Select high-fidelity KOD-Plus-Neo DNA polymerase, PCR amplify HSV1-tk gene from pORF-HSV1tk, introduce required NotI and SfiI restriction enzyme sites, upstream The primers and downstream primers are 5'-GCAGGCCCGATCGGCCATGGCCTCGTACCCCGGC-3' (underlined is the introduced SfiI restriction site) and 5'-GCATCAGCGCGGCCGCTCAGTTAGCCTCCCCCAT-3' (underlined is the introduced NotI restriction site), and the reaction conditions are 94°C. 5min pre-denaturation; the following cycle 30 times: 98°C for 10s, 62°C for 30s, 68°C for 1min; then 72°C for 5min for final extension; finally, store at 4°C, amplified fragment 1130bps, after PCR amplification, 1% agarose gel Electrophoretic separation, the conditions are: voltage 120V, time 25 minutes; confirm the band size under ultraviolet light as image 3 , cut off the target band with a clean blade, recover and purify the DNA of the target fragment by gel, detect the recovered DNA concentratio...
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