3-6.2.3 In-vivo site directed mutagenesis

In vivo genome manipulation through site-directed mutagenesis can be done by various methods described as below.

a) In vivo site-directed mutagenesis with synthetic oligonucleotides:

This system combines the versatility of synthetic oligonucleotides for targeting with the practicality of a general selection system. It provides for an enormously wide variety of genome modifications via homologous recombination. Exceptional high frequencies of mutations can be obtained when a site-specific double-strand break (DSB) is induced within the locus targeted by the synthetic oligonucleotides. (Storici F et al, 2006 )

b) Trans-placement "pop-in pop-out"

Most gene targeting experiments have been used to disrupt endogenous loci, resulting in targeted null alleles (strategy is often termed as ‘gene knockout'). Two types of vector have been developed for this purpose: insertion vectors and replacement (or transplacement) vectors.

Insertion vectors are linearized within the homology region, resulting in insertion of entire vector into the target locus. This type of vector disrupts the target gene but leads to a duplication of the sequences adjacent to the selectable marker.

Replacement vectors are designed so that the homology region is collinear with the target. The vector is linearized outside the homology region prior to transfection, thus crossover events in which endogenous DNA is replaced by the insert DNA. With this type of vector, only sequences within the homology region (not the vector backbone) are inserted. Thus for gene knockout, the homology region itself must be interrupted. Insertion and replacement vectors

are equally efficient, but replacement vectors have been used in the majority of knockout

experiments.

c) Direct gene deletion and site-specific mutagenesis with PCR and one recyclable marker using long homologous regions.

Several methods have been developed till now to obtain a successful mismatch mutation (transition/ transversion) in the targeted site. Some of them are discussed below:

3-6.2.4 Spontaneous hydrolysis

DNA is not entirely stable in aqueous solution. Under certain physiological conditions glycosidic bond may be hydrolyzed impulsively and thousands of purine sites in DNA are estimated to be depurinated daily in a cell. Several DNA repair pathways exist for the DNA, however, if apurinic site is irrepairable, mis-incorporation of nucleotide may take place during replication. Adenine is incorporated by DNA polymerases in an apurinic site. Cytidine may also deaminated to uridine at lower rate of depurination and can form G to A transition. Eukaryotic cells also contain 5'-methylcytosine, may be involved in the control of gene transcription, which can become deaminated into thymine.