11.1 Gene disruption
Yuji Hiwatashi and Tomoaki Nishiyama
1.
Strategy
This
chapter explains a brief outline for gene disruption with gene targeting
technique. A coding region of a targeted gene should be fully deleted from its
genomic locus to generate a null mutant. A mere insertion of a selection marker
in a coding region may cause an expression of a truncated gene. Polyadenylation
signals of the CaMV 35S and the NOS terminator in the marker cassettes do not
completely terminate transcription, and "read through" from the
marker cassette happens. When a specific domain of a gene is crucial for its
function, complete deletion of the domain will be sufficient for loss of its
function.
To fully delete a coding
region, genomic DNA fragments 5' and 3' the coding region are required for
construction of a targeting vector. The genomic DNA fragment is recommended
to be longer than 1 kb. The corresponding genomic fragments are usually found
in DNA data blase (see 10.0).
2.
Construction of a targeting vector
The genomic fragments
corresponding to homologous regions are inserted into 5’ and 3’ sides of a
selection marker cassette, respectively. The pTN182 and pTN186 vectors confer
the resistance to G418 and hygromycin, respectively. The PCR fragment with
blunt ends, which is amplified with proof-reading DNA polymerate such as KOD
plus DNA polymerase (TOYOBO), can be directly cloned in the EcoRV and SmaI sites of
those vectors.
(caution for primer design when genomic DNA sequence is not
available and you need to use TAIL-PCR)
Arrange primers to leave
genomic regions for later PCR screening of transformants. Most right and left
primers will be arranged for transformant screening in the following figure.
3.
Transformation and selection of candidate lines
The constructed DNA
fragment for targeting is excised from the vector with appropriate restriction
enzymes. Purification of the fragment is not necessary, and we usually use a
mixture of the DNA fragment and a vector for transformation. Approximately 10 µg
of the DNA fragment-vector mixture is introduced to protoplasts. Lower and
higher DNA amount cause less transformants and more insertions, respectively.
Approximately 5 – 10 µg PCR amplified DNA fragment is possible to
use for transformation.
PCR and Southern blot
analyses are used to select transformed lines with a correct replacement. PCR
is used at the first stage of screening, and then southern analysis is
performed. To avoid an unexpected recombination, you definitely need to perform
southern hybridization before proceeding to further analyses of the
transformants.
(Three kinds of PCR
reactions are recommended) see figure below.
(1) Use
primers that anneal to 5' genomic region and to a 5’ side of a selection marker
cassette.
(2) Use
primers that anneal to 3' genomic region and to 3’ side of a selection marker
cassette.
(3) Use
primers located in the selection marker cassette to eliminate a line with
tandem insertions.
