Note for Reduced X-Ray Resistance and Homologous Recombination Frequencies in a RAD54(-/-) Mutant of the Chicken DT40 Cell Line

It is very old paper though -

Note for: Reduced X-Ray Resistance and Homologous Recombination Frequencies in a RAD54(-/-) Mutant of the Chicken DT40 Cell Line

(doi: 10.1016/S0092-8674(00)80198-1)

Done in S. cerevisiae before and want to investigate in vertebrate -- therefore, DT40 is chosen.

1. Rad54(-/-) was generated

2. Testing this KO - found out that sensitive to radiation, Ig gene conversion reduce, lower targeted integration

3. Complementary testing with human cDNA -- regain radio-resistant and targeted integration

**first report on DBS in vertebrate cells.

In yeast model study --> found out that either Rad51,Rad52 and Rad54 mutants --> DSB stay long after injure by X-ray

Targeted integration in mammal is hard due to the majority of DSB repair occurs through NHEJ (my guess would be less S-phase period and no check point during intra-S? --> need to check which mammalian cell lines were used)

Vertebrate cell line model:

DT40 is a good model to study homologous recombination in vertebrate.

Advantage of this cell;

1. Still be able to diversify its Ig light chain like its appearing in bursal B-cell

2. Increase ratios of gene targeting

Point of this paper;

First report on Rad54 KO phenotype in vertebrate cell line.

Profile of Rad54 -chicken

1. 742 aa

2. share identity 83% and 48% AA with human and S. cerevisiae

3. mRNA expression level by northern blot -- highly in bursa thymus, testis, ovary and lower in other organ tested

The construct was designed to delete the aa from 318.

1.Rad54(-/-) -->viable but grow slow -->mean this gene is not essential for proliferation.

2. Observe from the second allele KO -->Rad54(-/-) clones have declined survival rate.

Targeted results showed;

1. 1st allele KO -- 55% total clone

2. 2nd allele KO -- 7% total clone

meaning -- Rad54(-/-) clones have declined survival rate.

Analysis of Ig-light chain conversion rate can indicate the function of targeted integration.

Functional study done in yeast and chicken were similar --> therefore, the function of this gene is conserved along the eukaryotic evolution.

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