Final answer:
Creating a conditional transgenic knockout involves engineering an sgRNA, cloning it into a CRISPR/Cas9 array on a plasmid, transforming cells, and using the CRISPR system to target and mutate specific DNA sequences. This is often followed by processes such as agarose gel electrophoresis, DNA cleanup, and tissue culture to select successfully modified cells.
Step-by-step explanation:
To create a conditional transgenic knockout, scientists follow a series of steps. They first engineer an sgRNA with the desired mutations targeting specific sites in chromosomal DNA. Then, this sgRNA is cloned into the CRISPR/Cas9 array on a plasmid. After the plasmid is introduced into cells through a process called transformation, the CRISPR/Cas9 system forms a complex with the target DNA sequences within these cells. The enzyme then creates nicks in both strands of the target DNA at the specified location. The cell's own DNA repair mechanisms can introduce mutations at the site of the cut, which can lead to a 'knockout' of gene function, conditional on the presence of certain factors that control the expression of the CRISPR/Cas9 system. These factors could include tissue-specific promoters, inducible systems, or chemical controls that allow for precise spatial and temporal regulation of the gene knockout.
In the laboratory, the process might begin with the digestion of plasmid DNA using a restriction enzyme to create a linearized form that can be inserted into the host cells. Agarose gel electrophoresis is then used to evaluate the success of this reaction. Additional steps such as phenol-chloroform extraction and ethanol precipitation may be used to clean and concentrate the plasmid DNA before it is introduced into cells. This is followed by tissue culture and selection processes where cells that have successfully integrated the transgenic construct are identified and propagated.