Re: How do they get the CF gene into the adenovirus in Gene Therapy?

I have included the following answer from the Mad Sci Archives in case you 
missed it. 


An electroporation machine uses electricity to make tiny holes in the cell 
wall. These holes are temporary so the procedure must be done quickly. See
http://www.cytopulse.com/portut.html
 .

Electroporation can be used in eukaryotic cells and bacteria to introduce 
DNA into the cells. The cells are then infected with adenovirus. At a 
certian point in the cell cycle, the virus starts packing the genetic 
material into the virus shell, or "capsid".  If a cell has DNA introduced 
into the target cell and the cell is then infected with the adenovirus the 
virus will package the plasmid DNA as if it wee it's own a certain 
percentage of the time. One can then select out the virus of interest and 
use it for further study, like the one below. This technique is less 
sophisticated that the one described below but it has been used in the 
past. 

Also see:  http://esg-www.mit.edu:
8001/bio/rdna/cloning.html


I hope this helps,
James
	
Area: Genetics 
Posted By: Sharon Shriver, Instructor (faculty; Ph.D.), Dept. of 
Pharmacology (I do molecular
genetics), University of Pittsburgh
Date: Tue Sep 2 10:03:35 1997
Area of science: Genetics 
ID: 871525196.Ge 

Message:

Dear Wayne,

        Gene therapy is one of the most interesting and valuable techniques 
to 
come out of the field of genetic engineering.  The adenovirus used to 
deliver the CF gene to human cells for gene therapy is known as a vector.  
Since most cells normally will not take in or absorb DNA, we need a 
delivery system to get the gene into the cell.  Adenovirus is a good vector 
since it can infect cells in vivo, or while they're in the body, which 
means that the gene can be delivered through an inhaler (instead of having 
to manipulate the cells in vitro, or in the laboratory, then return the 
cells to the body).  To use the adenovirus as a vector, it's genome was 
first altered by removing all the virus DNA except for the minimum 
necessary for the virus to live and infect the cells.  Genetically 
engineered viral vectors like this are harmless and usually can't live 
outside of the laboratory.

        A clone, or copy, of the CF gene was then inserted into the viral 
genome, or what was left of it, so that the virus thinks the CF gene is 
part of it's own DNA.  We can "cut and paste" DNA in this way using enzymes 
which have been purified from other viruses and bacteria.  Restriction 
enzymes cut (or restrict) DNA at specific DNA sequences, and ligases attach 
free DNA ends together.  In this way the unwanted genes are cut out of the 
viral genome and the CF gene is pasted in.  The viral genome would then be 
called recombinant.  We then use other purified proteins and enzymes to 
build a new virus, or "package" the DNA.  To do this, the recombinant DNA 
is mixed with all of the protein parts of the virus (for the outer coat, 
and the parts that attach to the cell, for example) along with some viral 
enzymes that assemble the virus, and in the test tube a whole, intact virus 
is created with the new DNA inside.  After infecting some cells in the 
laboratory, the new virus makes millions of copies of itself (carrying the 
CF gene), which can be purified and used for gene therapy.