plasmid rescue method - (Jan/31/2006 )
does anyone know the mechanism of plasmid rescue method? i read a paper in which this method is used , but i can not understand it well.
Hello,
Basically, say you use homologous recombination to mutate some gene in a bacterium. Let's say this gene is responsible for normal cell morphology and, without it, the cell assumes a cube shape. Now, let's say you transform this mutant, cube-shaped cell with a plasmid that has a normal copy of the gene mutated earlier. This plasmid also has a constitutive promoter cloned upstream of the wild-type gene, so the gene is constantly being expressed. The cube-shaped cell now has a copy of the gene responsible for normal cell morphology (and thus the proper gene product), and so the cell should lose it's cuboidal shape and revert back to a wild-type phenotype.
Many researchers use plasmid rescue to verify gene knock-out experiments. If you knock a gene out and observe something abnormal, then re-introduction of that gene should "rescue" the original state of the cell, hence the term.
I hope that makes sense. I've been writing all day and I'm a bit burned out!
-Hank
Here's an example of a good paper that uses "plasmid rescue" to demonstrate a principle (i.e. tat pathway is essential for this process):
http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum
Basically, say you use homologous recombination to mutate some gene in a bacterium. Let's say this gene is responsible for normal cell morphology and, without it, the cell assumes a cube shape. Now, let's say you transform this mutant, cube-shaped cell with a plasmid that has a normal copy of the gene mutated earlier. This plasmid also has a constitutive promoter cloned upstream of the wild-type gene, so the gene is constantly being expressed. The cube-shaped cell now has a copy of the gene responsible for normal cell morphology (and thus the proper gene product), and so the cell should lose it's cuboidal shape and revert back to a wild-type phenotype.
Many researchers use plasmid rescue to verify gene knock-out experiments. If you knock a gene out and observe something abnormal, then re-introduction of that gene should "rescue" the original state of the cell, hence the term.
I hope that makes sense. I've been writing all day and I'm a bit burned out!
-Hank
Thank you very much for your reply. the basic mechanism seems like what you said. however the paper i read seems more complicated. i copied the experiment process under. their purpose is to gain the sequence of genomic in which the plasmid has been inserted.
Plasmid rescue was done as follows:
Briefly, genomic DNA was extracted
from ES cell clones with lysis buffer
(0.1 M Tris-HCl pH 8.0, 5 mM EDTA,
0.2% sodium dodecyl sulfate, 0.2 M
NaCl with 0.2 mg/ml of proteinase K).
Ten micrograms of DNA was digested
with NcoI overnight, cleaned, and ligated
at a concentration of 5 g/ml.
The reactions were incubated overnight
at 16°C. Transformation of
DH10B (GIBCO) was done by electroporation
with 1 g of ligated DNA.
Colonies were selected for ampicillin
resistance, and the rescued plasmids
were sequenced. In addition to the
plasmid rescue method, DNA Walking
SpeedUp Kit (Seegene) was used for
cloning of the transition site between
the gene-trap vector and genomic
DNA according to the manufacturer’s
instructions.