Transcript Lec15-Recombinant

Recombinant DNA
Scope
Human Genome = 3x109
Average Gene = 3x104 (1/105)
SNP Mutation (1/109)
Process
Cut DNA into pieces
Insert DNA into vectors that can replicate in bacteria
Transform (introduce) DNA into host cell
Plate cells and select those with vectors
Each colony has one chunk of DNA
The whole set is a library of human DNA
Specifics
Restriction enzyme GAATTC
Palindrome
Exists for a large number of loci
Ligase can bind together DNA
from any organism that has
been cut with the enzyme
Comes from bacteria that need
to repair their own DNA
Hook fragment to bacterial DNA
with a replication site.
Selecting Cells
Episomes
Contain resistance genes
Are transferred between bacteria to allow for more
rapid adaptation
We can insert all of the fragments from the human
genome into bacterial episomes and create a library
of bacterial colonies that can be used to replicate
DNA
Reverse Translation
You can take a protein like beta-globin and determine
the amino acid sequence
You can then create a set of complimentary probes
for all possible nucleotide sequences that would
cause the amino acid sequence
Dump the probe on each entry in the library to find
the gene that makes beta-globin
Other Approaches
Insert DNA into a virus and infect malalian cells.
Create artificial chromosomes and inject in the
nucleus
Bacterial Artificial Chromosome
Stem Cell Manipulation
Lets grow insulin
Insert insulin gene into plasmid
But promoters for e-coli are
different than human
So put e-coli promoter on the
front of episome
But humans do splicing and
bacteria don’t
So, we need to insert spliced
gene into epiisome
Use reverse transcriptase to
turn RNA into DNA
Viruses use this to replicate
But protein may fold differently
in bacteria, so you may have to do
some more engineering
How about mammals
Rabies antibodies are important
It is hard to find people that generate them
People don’t like to be infected to generate antibodies for pharma
companies
Put human gene that generates antibody into mouse, infect it and
collect antibodies
But mice don’t have much blood, lets do it with a cow
But the cows immune system also generates cow antibodies
So lets cut out the cow immune system genes and totally replace them
with human immune system
Hope that the cow doesn’t get sick