Note for: Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40

Note for: Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40

(doi: 10.1098/rstb.2000.0755)

DT40;

- transformed B-lymphocyte cell line

- infected with leucosis virus

- high targeted/random ratio

- result from the property of cell itself which has diversification properties to produce various Ig through DNA recombination

- Making this cell line easy to generate the gene-targeted mutant

Strong similarities phenotype between DT40 and murine of gene involving in DNA "recombination"

From theirs phenotype similarities --> suitable for studying DNA "recombination"

Gene-targeting normally done in;

1. murine embryonic stem cell

2. human cell lines

efficiency is normally low-->10^-2-10^-5

Reason behind why DT40 having high targeted/random integration is unknown. There are three from chicken B-lymphocyte cell lines which have high targeted integration; two avian (ALV)-transformed cell line DT40, RP9, and v-rel-transformed cell line 27L2.

Other than B-cell line-->no targeted integration.

It has been postulated that the higher ratio of targeted-->may relate to Ig gene conversion activity-->the activity processes through the HR which only B-cell has this kind of activity.

Advantage,

1. high targeted ratio

2. tractability -- stable phenotype and karyotype

Chicken chromosome;

80 chromosome -- 11 autosomal macrochromosomes, ZW sex chromosomes and 67 microchromosome

Uniqueness of this cell line;

trisomy at chromosome 2

one additional microchromosome

other than that the karyotype looks fine.

Therefore, when the targeted gene is disrupted, the effect should come from the disappearance of that gene rather than the variation between clone by clone.

Three methods are normally done with conditional (bc DNA repair is important process for living cells - dysfunction of the key genes could cause cell dead and rarely to study the phenotype);

1. tetracycline repressible promoter -- leaky expression

2. Cre recombinase - LoxP system -- not occur synchronously comparing with the tet (randomly occur when introducing the Tamoxifen)

3. Generation of temperature-sensitive mutants--DT40 cell can grow from 34-42.5C-->must design based on the yeast ts mutants.

To determine HR between sister chromatid --> SCE can measure such that-->it involved with S-phase associated repair-->also to study this we can treat the cells with mutagens prior to DNA replication.

Normally, cycling mammalian and chicken-->5 spontaneous SCE/mitosis.

HR responsible for both spontaneous SCE/cross-linking SCE.

Gene-targeting efficiency--relies on homology length, which increased from 6-12 kb. But in the lower eukaryote like yeast, requires shorter homologous sequence to get good efficiency (fewer than 100 bases as mentioned in this paper).

the relationship between each repair pw. in yeast and vertebrate cells works differently.

Yeast, HR-mediated repair can be found any state of cell cycle. HR-related genes are induced after genotoxic treatment.

Vertebrate, HR-mediated repair -state specific and genes related to the process like Rad51 and Rad54 are not expressed in resting state, even though, the cells have been exposed to genotoxic treatment.

HR is the pw for fixing spontaneously occur DBS break during replication whereas  NHEJ is for fixing DSB which induced by IR.

HR is important for maintain genome integrity while NHEJ is very minor-->this is in the case of error in the replication.

Cell cycle checkpoint plays more role in vertebrate cells rather than in yeast.

HR system interacts with cell-cycle and checkpoint regulation--unknown

all these interactions in yeast cannot be implied in vertebrate cell.

Two action modes (above figure):

1. targeted construct is linearized and the intact genomic template in the cell is used to initiate HR. (yeast)

2. targeted construct participate HR-mediated upon the genomic DNA has been initiated for HR (through the damage) - vertebrate case

the later one seems to increase the efficiency of gene-targeting.

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