Inaktivasi DNA dalam sel

mamalia
Afifa Khairinnisa
Human somatic cell gene targeting
• Provides a powerful tool to scientists studying
gene function in cultured human cells.
• importance, Because it can be used to either
delineate the loss-of-function phenotype of a
gene or correct a mutated gene back to wild-
type
 Strategic planning
• Selection of parental cell line
Six different human cell lines have been
successfully used for human somatic cell gene
targeting
Cell line : HCT116, DLD1, HT1080, SW48, HaCaT,
Human fibroblast
 Strategic planning
• Selection of parental cell line
Six different human cell lines have been
successfully used for human somatic cell gene
targeting
Cell line : HCT116, DLD1, HT1080, SW48, HaCaT,
Human fibroblast
Architecture of promotorless targeting
vector
• Promotorless targeting vectors require that
the selectable marker gene be inserted in-
frame into the gene being targeted.
• There are several general factors to consider
when planning to construct such a targeting
vector
 Building a targeting vector
• Building promotorless targeting vectors is a
nontrivial exercise in recombinant DNA
technology
• 2 factors:
• There is virtually no sequence flexibility in the
juction between the left arm of the targeting
vector and the selectable marker cassette
• Second, it is frequently necessary to add
several sequence features between the end of
the selectable marker gene and the right
homology arm and targeting vector,
• Such as retriction sites and a PCR priming site
Screening for Knockouts in human cell
• The simple approach for identifying gene-
targeted cell line is to conduct an initial-PRC-
based screen
• Then confirm the putative knockouts with a
Southern blot
• Two general factors
 Targeting the second Allele
• One particularly challenging aspect of human
somatic cell gene targeting is the creation of
homozygous knockouts by disruption of the
second, remaining allele