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CMB1003 L16 Exam Study Guide
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CMB1003 L16 Exam Study Guide ...
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CMB1003 L16 Exam Study Guide useful properties of viruses we can exploit - Answer -natural gene delivery vehicle trigger immune response many virsuses have cytopathic effect -virus are easy to modify/manipulate at genetic level to make recombinant virus -target delivery to specific cells -powerful gene expression -can carry foreign protein genes -can remove components which make a virus harmful attenuated genes - Answer -human viruses that have lost the properties to replicate efficiently in human cells -not disease causing -non human viruses that can infect human cells, limited replication if any but some gene expression gene expression powerful promotors/enhancers - Answer - virus or elements of viruses have been used to express many cellular protiens -human cytomegalovirus promotors/enahncers -simian virus 40 -HIV long terminal repeats common laboratory gene expression - Answer -promotor region from a virus which has been cloned into a DNA plasmid vector: gene expression vector, modern ones have multiple restriction enzymes sites for ease of cloning, with polyadenylation sequence, and may have splice sites •Generate cDNA of protein you wish to express from mRNA (no splice sites) using PCR Clone into vector Propagate in bacteria Isolate/purified transfect into human ce pcDNA3.1 - Answer Pcmw= promotor ampicillin and pUC ori allow replication and selection in bacteria neomycin and sv40 ori allow replication and selection in human cells transfection - Answer isolate ORF of interest clone into expression vector isolate purify plasmid DNA transfect target cells and detect gene expression efficiency can be variable, hard to hit all/most the cells in a culture disk the use of viruses as vectors - Answer clone into virus/ VLP (virus like particle) -allow more efficient delivery than chemical/liposomal transfection -using own virus entry mechanism -infect hard to transfect cell lines adenoviral vectors - Answer •A common vector for gene expression in research laboratories •Is the vector used in deliv ering Covid vaccines pros: small genome, easy to work with, easy to purify, infect many cell types, high level of gene expression Disadvantage •Limited to ORFs <8 kb •Immunogenic, means single shot •Transient expression Adenovirus replication - Answer virus fibers attach to host cell receptor penetration by endocytosis into endosome uncoating- release of virionThe benefits of buying summaries with Stuvia:
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