Re: How does gene splicing work?

   Hi Robert. Your question really refers to gene therapy, in which a 
gene can be introduced into a patient who presents with a genetic 
disorder.  I will focus on that.
   First, in order for gene therapy to be performed, the gene corresponding
to the abnormal gene in the patient must be identified and spliced into
the appropriate therapeutic vector.
   Identification of genes involved in hereditary disorders is usually
accomplished by looking for co-inheritence of specific chromosomal
genetic "landmarks" with the disease in family trees.  These genetic
landmarks bring the investigator to the general area of the mutated gene.
This is like trying to find someone in Chicago by looking for the Sears
Tower- you know it's Chicago and that the person you're looking for is
somewhere in the general vicinity.  Now it's time to do a street-by-street
search.  
   Once the gene has been identified, it is spliced into a bacterial plasmid.
The plasmid is a circular piece of DNA that replicates over and over in
bacteria, producing hundreds of copies of itself in each cell.  Large
amounts of the spliced gene can therefore be made in the bacteria and 
then purified in the test tube.  In general, splicing is accomplished by
digesting the DNA with one or more enzymes(restriction endonucleases) that
cut DNA at specific places (determined by the presence of specific
nucleotide recognition sequences). Another enzyme (DNA ligase) sews the
DNA fragments into a bacterial plasmid that is added to the mixture.  This 
plasmid, containing inserted human DNA, is then driven into bacteria where
it is able to replicate. 
   Finally, the gene is respliced into a therapeutic vector.  The vector
used depends on the disease.  For instance, cystic fibrosis is treated 
with a therapeutic vector that is transported into the patient's lung
cells in the form of a modified virus (like a cold virus) that is able to
infect lung tissue. The viral DNA is injected into lung cells which need 
the normal version of the cystic fibrosis gene in order to function properly.  
Since the viral DNA contains a spliced cystic fibrosis gene that acts 
normally, the symptoms of the disease are alleviated.

-Dan Wexler