Note for: Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40

Note for: Reverse genetic studies of the DNA damage response in the chicken B lymphocyte line DT40

(doi: 10.1016/j.dnarep.2004.03.039)

Post-genomic era -->

1. reverse genetic tool is used to study the protein function

2. ppl using DT40 bc of many advantages;

- efficient gene targeting

- stable phenotype

- lack p53 allow us to study the DNA damage due to the cells still can go through the interphase checkpoint even they have the error --  this is the reason why it is a good model to study the DNA damage and repair by observing on the chromosome.

In higher eukaryote;

1. human and mouse, B-lymphocyte develop in bone marrow

Functional IgV segments generate through site-specific process called V(D)J recombination

2. rabbit and chicken, B-lymphocyte develop in specific tissue; R-appendix of intestinal tract/C-bursa of Fabricius

Functional IgV generate through intragenic homologous DNA recombination, called Ig gene conversion.

Mature B-lymphocyte;

human and mouse -- supply from bone marrow throughout life

chicken -- self-renewal w/o new supply from primary organ

Affinity maturation also different;

human and mouse -- accumulation of base substitution after being stimulated by antigen

chicken -- Ig gene conversion, then antigen stimulant, diversify IgV through single base substitution

DT40;

- transformed by avain leukosis virus

- Ig gene conversion always

- Good targeted integration

It has been observed that good gene targeting is unique property of chicken B-cell lymphocyte.

Like mentioned in a few previous publications; molecular mechanisms why chicken B lymphocyte has high gene targeted is unk, but they propose it may be related to Ig gene conversion (this process occurs through HR, and lymphocyte lines have this).

Construction of large plasmid will be troublesome.

For the database;

1. chicken database

2. chicken EST (http://www.chick.umist.ac.uk)

3. DT40 EST (http://genetics.hpi.uni-hamburg.de/dt40.html) -- not working anymore

For the mouse ES (when generating KO line) - the length of similarity sequences required 10 kb, whereas, in DT40 requires 1.5-3 kb, in yeast it even shorter.

Another factor which affect the gene targeting efficiency; the length in genomic being disrupted; 110kb and 57 kb delete -- 1/56 and 1/48 (larger area occur harder)

Reverse genetic study advantage;

1. stable karyotype and phenotype even extended periods of culturing, whereas, mouse ES - multipotency is lost while passaging and when being exposed to genotoxic stress. Besides, multiple genes KO could be performed by using different selectable markers (neo, his, bsr, his, ecogpt, and zeocin) -- allow us to see the effect of more than one gene.

2. rapid growth rate, at 40C - 8-10 hr for doubling time; therefore, it is easy to study the phenotype on cell viability and proliferation, like colony formation assay, etc.

3. cloning efficiency of WT is nearly 100% - isolate the stably transfected cells and subculture is easy

4. lack of p53 (induce apoptosis and cell cycle arrest in G2 phase - after damage) -- allow us to study the gene that maintain the genome instability, whereas in mammalian cells could not be performed (lethal). Bc of its resist to the apoptosis, also allow us to study the extent of genome instability through spontaneously arising chromosomal breaks in mitotic cells (passing through G2 due to the lack of checkpoin activity of p53).

Four method to generate the conditional KO;

1. Targeted KI by which using the LoxP/Cre or Ts transgene - generated heterozygous first and introducing the system that can be inducible-off (inducer can be Cre-recombinase, temp)

2. Tetracycline inducible promoter

- using tetracycline to promote the expression of transgenes

- they construct the rescue plasmid cassette which contains tet-inducible promoter+transgene+IRES+luciferase gene to access the level of expression.

- few technical problems, leaky expression, but can be fixed by looking at the luciferase activity and pick the clone with the lowest activity/or using the tet-repressor to suppress the leak

- another issue, tet promoter is strong promoter, overexpression beyond the physiological level - some phenotype could not be tolerate and requiring the screening before further experiment.
- another drawback; slow reduction of transgene; hard to interpret the results from a null phenotype

3. Cre-site specific recombinase; gene that flanked by loxP will be deleted upon the TAM incubation with the plasmid MerCreMer (Mer -- mutated estrogen receptor)

- Cre/loxP clean system to stydy the null phenotype but tet having the leaky problem; therefore, it Cre/loxP is perfect to study synthetic lethality

- disadvantage; Cre/LoxP not synchronous but tet works synchronously

- Cre recombinase + TAM are toxic to the cells

- the absence of cre-mediated deletion in a small fraction of cells though TAM is continuously incubated

4.chimeric protein with ER (estrogen receptor) and Ts

- some phenotypes are quantitatively sensitive, in this case, they gave the example of CENP-C (structural components of centromere) which rescue transgene could not be used since it produces a lot of protein. The other methods, like tet, cre/loxP, and fusion ER ---> did not give sudden inactivation--> hard to interpret the result since there might be some interfered factors (I guess)

- therefore, they make use of temperature of this cell line to study the phenotypes. They generate the mutant of CENP-C which is in the temp-sensitive form (require database to design). Since DT40 is able to grow in the wide range temp; 34-43C, viability is still the same. As a result, when culture the DT40 with ts version, the cell will show the phenotype if that protein has the effect on viability, whereas at 34C, the cell is still grow normally.

Phenotypic analysis of essential DNA repair gene;

cause and effect --> lost of DNA repair --> accumulation DNA damage --> stimulate the damage checkpoint --> apoptosis if to much injured!

Authors mentioned that DT40 cells can carry a few chromosomal breaks and go through metaphase w/o activation of G2-checkpoint. Single chromosomal break in mitotic chromosome --> single unrepaired DSB -->cell death in the end.

Therefore, we can study the targeted genes (through KO) which believed to involve in DNA repair by observing on the chromosomal break in DT40.

Authors also point out that yeast genome is not large so it is hard to observe the chromosomal morphology which can be used to study the genome maintenance/integrity.

**Assay used for measuring HR in DT40

1. intragenic HR induced by I-Sce-I restriction enzyme --> has been used in mammalian system and it is good to differentiate between HR that occurs from either intragenic or sister chromatid

2. intragenic HR in Ig gene conversion --> besides assessing the HR, it also determines the base sequence of HR products which can represent the fidelity of HR.

3. Rad51 focus formation after IR

4. Gene targeting efficiency

5. Sister chromatid exchange (SCE)

6. Sensitivity assay challenged by IR, crosslinking agents

Assay from 3-6 --> author mentioned that there are the other factors that interfere with HR besides the targeted gene that we investigated.

7. spontaneously arising chromosomal break

Example of severity for G2 analysis after IR (only DT40);

1. super severity -- Rad50, Rad51,Mre11, Nbs1, Brca2, Rad52 and XRCC3

2. fair severity -- Rad51 paralogs

3. less severity -- Rad52 and Rad54

Sensitivity by genotoxic agents -- many factors involve;

1. various DNA repairs

2. damage checkpoint

They gave the example of RAD18-KO which showed sensitivity toward diverse kinds of genotoxic agents --> hard to say which repair pw is defected through these genotoxics analysis.

IR sensitivity toward "synchronized populations" of cells is informative to determine the impaired repair pw.

They mentioned that both "mammalian and DT40" tolerate the IR at the late S-phase but not the early S-phase (no sister chromatid) --> if the gene -disrupted clone shows the sensitivity during the late-S phase after IR --> reflect this gene play role in HR-mediated DSB repair.

Another approaches to investigate the HR by IR in G2 phase --> measuring chromosomal breaks in the following M-phase --> if there are the severed phenotypes like chromosomal break or cell death --> that disrupted gene is related to the HR. Besides, the level of severity also determines the relative distant of the disrupted gene.

**NHEJ -- key proteins

1. Ku

2. DNA-PK

3. XRCC4

4. Lig4

HR play more role during replication (S phase since) but NHEJ play more in G1 phase.

The authors also mentioned the different between DT40 and mammalian cells in DSB repair --

HR is preferentially used in DT40 cells but HR and NHEJ might contribute equally to DSB repair in mammalian.

Phenotypic assay for NHEJ in DT40;

1. NHEJ-deficient cells showed extreme IR-sensitivity at G1 phase but not G2

2. Using linearized plasmid DNA, that contain directed repeat at the ends -- examine the microhomology or no homology activity (require Ku70 and DNA-PK) to create recircularization.

3. Disruption of Ku70 in gene disrupted cells -- inform about whether the gene use the NHEJ, for instant.

Author also suggested assay on VDJ-recombination might be informative based on the base sequence analysis of VDJ joints.

**Translesion DNA synthesis (occur with the cycling cell)

 - replication block will be released by post-replicational repair (PRR); HR and TLS

- three classic phenotype assays f TLS in budding yeast; 1. using the duplex plasmids carrying adducts (lesion) within GGG or GGCG sequence in one strand  and analyze based on the replication of each strand, 2. determination of induced mutation frequencies after exposing the cells with mutagens -- observe the base substitution which caused by TLS pw. (in yeast -- polZ and Rev1p are used to generate the spontaneous mutation), 3. measuring the size of newly replicated DNA before and after UV radiation. All 3 assays have not been developed in DT40.

Prominent phenotype of TLS-deficient DT40;

1. extremely high sensitive to crosslinking agents like CDDP

2. TLS mutants derived from DT40 and murineES -- Rad18, rev1,polk, polz have elevated sensitivity toward diverse kind of genotoxic agents.

3. increase in UV-induced chromosomal breaks, esp. when exposing to UV at early S-phase

4. increased spontaneous as well as induced SCE (defective in TLS caused the upreguation of HR-PRR)

Conclusion;

Why DT40;

Yeast is the good model to study eukaryote but there are some ortholog proteins which have discrepancy. For instance, Rad51, Rad2 and Rad54 mutants show the similar phenotypes in yeast, however, in mammalian, Rad51 is lethal and mice deficient in either Rad52 or Rad54 are viable and no developmental defect.

Genome instability affects the viability of murine fetus and primary cell culture. Though, if the mutant mice are viable, the relative primary culture often time will have limited life span therefore it is hard to study. Therefore, transformed cell line is the good choice to use. Let taking a look on DT40, the authors convinced that there were no significantly different phenotypes of HR-deficient mutants between DT40 and murine ES. Therefore, DT40 is a good cell line to use as reverse genetic tool to study the HR?

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