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Exam #3 Gene Therapy and Genetic Engineering Questions with Complete Answers
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Exam #3 Gene Therapy and Genetic Engineering Questions with Complete Answers
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Exam #3 Gene Therapy and GeneticEngineering Questions with Complete
Answers
Where does the gene sequence that I put into a plasmid come from? - Answer--A
sequence that has already been cloned and put into a plasmid using restriction
enzymes
-make cDNA primers with PCR
If the DNA is in a cloning vector, what must occur before it can be used to treat cells?
What about expression vectors? - Answer--it must be transferred to other vectors
-expression vectors are already ready to be used to treat cells
What are the methods for getting plasmid DNA into cells WITHOUT the help of viral
proteins? - Answer--Lipofection (liposome transfection)
-Electroporation
What are the steps in liposomal transfection of a cell? - Answer--DNA associates with
liposome
-Liposome and associated DNA fuses with the cell membrane (both membrane and
liposome are made of phospholipids)
-DNA is inserted into the cell
-Plasmid DNA is maintained extra-chromosomally
Why is gene therapy using liposomal transfection not ideal? - Answer--Is not integrated
into the chromosome, so not passed to progeny
-Very transient, would require a LOT of injections/treatments
What are the steps of electroporation? - Answer--Before the pulse, the cell is in a media
that contains the genes/drugs for transfection
-A pulse of electric field is introduced into the cell and causes a voltage across the cell
membrane allowing for the therapy to enter the cells
What are the advantages of using non-viral transfection mechanisms? - Answer--
relatively safe
-Large expression cassette delivery
What are the disadvantages of using non-viral transfection mechanisms? - Answer--
Transient expression
-Little transduction/low efficiency
, What are the overall steps/ questions that must be answered during the process of
liposomal transfection? - Answer--Find gene of interest (pubmed) -> is it a transcription
factor or a protein?
-Identify the region upstream of the start site and downstream of the stop site
-Identify what restriction enzyme you will use to cut it
-identify what vector you will clone it into(does the RE match the vector?)
-Is it a cloning vector or an expression vector?
-Identify the expression vector you will use to subclone it into
-Find reagents to transfect it
What can you add to cells? - Answer--Transcription factors (modulate expression of
existing genes)
-Proteins to over express (to treat deficiency/mutation)
-Proteins so I can tag/visualize a cell
What are the steps in CRISPR? - Answer-1. Scientists create a genetic sequence
known as a "Guide RNA" that matches the piece of DNA they want to modify
2. The guide RNA sequence is added to a cell along with the Cas9
3. Guide RNA binds to the target DNA
4. Cas9 enzyme binds and creates a double stranded break in the DNA
5a. Another piece of DNA (the target/desired sequence) is added to the cells and is put
into the genome in place of the old DNA by homology directed repair =new functional
DNA
5b. OR No additional DNA is added and the breaks are repaired with non-homologous
end joining =inactive gene
6. Enzymes repair the cuts
What occurs when a repair template DNA is present during the CRISPR process? -
Answer-Homology directed repair incorporates the new DNA that is designed to line up
perfectly with the cut DNA strands into the genome and creates a functional gene
What occurs when a repair template DNA is NOT present during the CRISPR process?
- Answer-Without a repair template, the ends of the DNA are joined by non-homologous
end joining, an error prone mechanism that does not perfectly replace the cut DNA,
leading to an insertion or deletion mutation that silences the gene = gene is inactivated
What are the steps of the mRNA transcription process in eukaryotes? - Answer--
Preinitiation complex assembly begins with the binding of the TBP to the promoter
(TATA Box)
-This binding causes a conformational change/distortion in the DNA
-TFIIh pries apart the double helix at the start point after positioning RNA polymerase 2
to start the process
What is the benefit of using viral vectors over nonviral vectors? - Answer-Improved
transduction/efficiency
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