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Location: Home > Custom Services > Molecular Biology Services > Recombinant Plasmid Construction and Gene Targeting

Recombinant Plasmid Construction and Gene Targeting

Date: 2018-01-25 Author: Leading Biology Click: 4588

Recombinant Plasmid Construction 



Recombinant Plasmids are the plasmid molecules which contain the target DNA fragment, and the process of introducing genes into a vector to form a new DNA molecule which can be replicated in a host cell is called Recombinant Plasmid Construction (Recombinant Cloning), the word “Recombinant” means that two different strands of DNA that would not normally occur together are combined and the cloning means creating multiple copies of genetically identical organisms.


Recombinant Plasmid Construction


There are five main steps in recombinant plasmid construction:
1. Target DNA extraction and PCR amplification.
2. PCR product purification.
3. Using restriction enzymes cut both plasmids and PCR products.
4. Connect the two loose pieces of DNA together using DNA ligase, form the modified plasmid.
5. Transform the modified plasmid into bacteria.

Gene Targeting


Gene targeting is the process of disrupting or mutating a specific genetic locus in embryonic stem (ES) cells, usually with the intention of making knock-out or knock-in mice. This method can be used for many proposes, including delete or insert mutations into genes of interest to determine their function; introduce human genes into the genome of mice to generate animal models enabling the study of human-specific genes and diseases; introduce individual genes or genomes of pathogens (e.g. viruses) in order to examine the contributions of such genes to the pathogenesis of the parent pathogens, or introduce reporter genes that allow monitoring in vivo or ex vivo the expression of genes of interest.


The design and construction of the DNA to be introduced into EC cells are critical and depend on the type of mutation required. The targeting constructs are usually a plasmid that contains two long stretches of genomic DNA, call homology arms, which are designed to match as closely as possible the genomic DNA of the ES cell line being targeted. These arms drive the homologous recombination event that results in the insertion of the construct into the desired locus.


Gene Targeting


The entire gene targeting process consists of the following major steps:
1. Linearize and purify the targeting construct.
2. Introduce the modified plasmid into ES cells by electroporation.
3. Grow clones under positive selection by antibiotics.
4. Pick the resistant clones and split them, extract DNA from those clones.
5. Genotype all clones by Southern blot using a probe specific for one end of the inserted DNA.
6. Expand clones that have the correct genotype. 
7. Inject the targeted ES cells into blastocysts, then implanted them into foster mice.



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