Chimeric vectors
Adeno-associated virus(AAV) is widely used in gene transduction experiments both in vivo and in vitro. AAV can infect a large variety of mammalian cells, producing only extremely low immunogenicity in the human body, and these characteristics make recombinant AAV a highly efficient viral vector used for gene therapy today. In the preclinical and clinical stages of gene therapy, recombinant AAV can be produced efficiently and large-scale by Baculovirus-AAV chimeric viral vector to meet dose and safety requirements.
Production of recombinant AAV using Baculovirus requires cotransduction of baculoviruses into insect Sf9 cells with both baculoviruses. The first baculovirus expresses the target gene located between the two AAV ITR elements, and the second baculovirus expresses the AAV rep and cap genes.For preparing two baculoviruses, the expression frame of each baculovirus was cloned into a baculovirus transfer plasmid. The entire expression frame of this plasmid and the Gentamycin resistance gene lie between the two Tn7 transposon terminal sequences on the plasmid (Tn7L and Tn7R).Therefore, the sequence between the two Tn7 on the transfer plasmid will be inserted into the lacZ gene after recombination by the Tn7 transposase and the rod, and the reconstituted E. coli can be obtained by gentamicin resistance screening and blue-white screening. Pole od extracted from E.coli were purified and used to transfect insect Sf9 cells.
This plasmid was transformed into E.coli with bacmid and the accessory vector. The bacmid can be viewed as a particularly large plasmid containing the modified baculovirus genome sequence. The sequence bears the lacZ gene and the attTn7 site located within the gene.When the host cell is transduced with AAV, the foreign DNA between the two ITR and the viral genome enter the cell together, and the linear double-stranded DNA genome exists in the nucleus in the form of free body DNA. In dividing cells, since free bulk DNA does not follow cell replication, the AAV genome will be progressively lost with cell division and therefore be diluted in the bulk cell.
The phenomenon of random integration of the host genome by DNA from recombinant AAV is very rare, which is quite applicable characteristics for gene therapy that needs to consider the insertion of exogenous DNA into the host genome to trigger an oncogenic risk.
Another advantage of AAV applications is that, in most cases, operations can be done in biosafety level 1 (BSL 1) facilities. Recombinant AAV is replication-defective and does not cause an inflammatory response and trigger human disease.
Many AAV virus strains have been identified from nature, dividing them into different serotypes based on different antigens of viral surface capsid proteins. Different serotypes have different tissue tropism (infectious tissue specificity).
Production of recombinant AAV using Baculovirus requires cotransduction of baculoviruses into insect Sf9 cells with both baculoviruses. The first baculovirus expresses the target gene located between the two AAV ITR elements, and the second baculovirus expresses the AAV rep and cap genes.For preparing two baculoviruses, the expression frame of each baculovirus was cloned into a baculovirus transfer plasmid. The entire expression frame of this plasmid and the Gentamycin resistance gene lie between the two Tn7 transposon terminal sequences on the plasmid (Tn7L and Tn7R).Therefore, the sequence between the two Tn7 on the transfer plasmid will be inserted into the lacZ gene after recombination by the Tn7 transposase and the rod, and the reconstituted E. coli can be obtained by gentamicin resistance screening and blue-white screening. Pole od extracted from E.coli were purified and used to transfect insect Sf9 cells.
This plasmid was transformed into E.coli with bacmid and the accessory vector. The bacmid can be viewed as a particularly large plasmid containing the modified baculovirus genome sequence. The sequence bears the lacZ gene and the attTn7 site located within the gene.When the host cell is transduced with AAV, the foreign DNA between the two ITR and the viral genome enter the cell together, and the linear double-stranded DNA genome exists in the nucleus in the form of free body DNA. In dividing cells, since free bulk DNA does not follow cell replication, the AAV genome will be progressively lost with cell division and therefore be diluted in the bulk cell.
The phenomenon of random integration of the host genome by DNA from recombinant AAV is very rare, which is quite applicable characteristics for gene therapy that needs to consider the insertion of exogenous DNA into the host genome to trigger an oncogenic risk.
Another advantage of AAV applications is that, in most cases, operations can be done in biosafety level 1 (BSL 1) facilities. Recombinant AAV is replication-defective and does not cause an inflammatory response and trigger human disease.
Many AAV virus strains have been identified from nature, dividing them into different serotypes based on different antigens of viral surface capsid proteins. Different serotypes have different tissue tropism (infectious tissue specificity).
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