HPRT mutation & 6-TG selection assay - (Jul/20/2012 )

Hello

got new cell line MRC-5 and MCF10A that can grow as monolayer in cell culture tissue flask.
I need to test their mutation frequency by treating with UV-A, UV-B, or UV-C radiation and I got a task from my boss to check HPRT mutation freq. in both cell line.

what I have done so far ...

First step ...
MRC-5 and MCF10A has plating efficiency ~ 20% and 70% for non-treated cells and I tested on 6-well plate.

Second step .... I have some problem ??
Use 6-Thioguanine (6TG) to select HPRT mutation cells, normal cell will die by taking up this 6TG ( from literature about 1 in 1 million to get this mutation in normal cells)
so I harvest 1,2 or 3 million in 100 mm² petridish

Poorly, I couldn't get any colony after treated with this 10-30 ug/mL 6TG ( I'm not sure how much concentration of 6TG that I should add to petridish)

I'm searching some proper protocol how to do HPRT mutation assay ....but couldn't find yet.
What I would like to ask is that if someone has any experience or some good protocol or any recommend or some articles/books?

It would be really grate appreciated ...

//Tai

It is quite possible that it hasn't been discussed before.

The concentration you used appears a bit too high (we use 5 ug/ml for our cell line). You have to do a killing curve using the cell line you are working with and then use the lowest dose that kill all your wildtype cells for selection.

Some protocols recommend that your wait for 8 days after introducing mutation before adding 6-TG.

you may also need to treat your cells with MNNG as positive control which is know to cause HPRT mutation and gives colonies on 6TG selection.

Here is a nice protocol on HPRT mutation assay found at http://depts.washing...cols_042009.pdf

this paper also gives a protocol" Frequency of HPRT gene mutations induced by N-methyl-N'-nitro-N

http://www.ncbi.nlm..../pubmed/9626969

pcrman on Mon Jul 30 15:47:09 2012 said:

Thank you for the protocol! I'm testing kill curve at the moment. I used quite high 6TG because my lung cells haven't been killed with below 10 ug/mL within 2 weeks. That why I did use extra high concentration in the beginning.

I have some questions, if HPRT- cells survive under 6TG medium. Will you see if they are forming like a single cell or forming like colony? As I tested so far, I can see only single cell spread everywhere on the cell culture flask with 6TG-medium after 14 days.


From the protocol, HPRT_protocols_042009.pdf as they mention about HAT supplement medium. I wonder if it is necessary to clean up all the TG resistant mutants before I start my experiment? If anyone know ?

Again, thank for the article, I will try with MNNG if it induce HPRT- on my cells.

Cells that survive 6-TG selection should form colonies, not exist as single cells. The colonies appear like the colonies you would see in antibiotics selection to establish stable cell line. A typical colony looks like the one in the image (d and f). Single cells are just dying cells which have been poisoned by 6-tg.

Hi
Thank you so much for clearifying with this images

After 2 month that I have tried with this HPRT Forward mutation assay, finally I could get colony. Wooohoooo!!

Here is my briefly protocol for Fibroblast, in case someone need!

First, Checking Killing curve with 6-TG some cell very sensitive some doesn't (try between 2-20 ug/mL)
Secondly, find the good positive control. ( I use UVC 0,48 j/m^2 and irradiated for 20sec to induce mutation)

1. Stock cells about 80% confluent, trysinizing and count number of cells about 3x10^6 cells
2. wash cell twice with HBSS without phenol red ( red color can absorb UVC light) in petridish 100mm
3. drop cells in petridish ( I use 3 mL cold HBSS, UVC can not penetrate HBSS if you put too much )
4. I put this petridish on Ice ( I have to run to UVC irradiator room about 5 min)
5. Irradiated with UVC ( MUST open the lid of petridish!!!)
6. quickly split to 3ea T175 cell culture flask (aprox 1million per flask but they will die during I run around with UVC. so I guessed that I have about 50% living cell left) and allow them to grow and subculture to add 6TG at day 3,7,11,16 after UVC exposure....keep them in 37C humidity incubator
7. for the day 3 I expand them ( safe some in 10% DMSO for future use)
8. day 7,day11,day16 I will subculture them and put 100,000 cells per 100mm petridish and immediately add 5ug/mL 6TG with cell medium and allow them about 14 days to form colonies!!
9. after two weeks, I will stain colonies with methylene blue in Methanol (4g/1L) for 10 min and then rince with water.
10. and see if how many colonies you have
GOOD LUCK

------------------
My results
My control ( with out UVC) -- 0 colonies
day 7 after UVC I got 4 colonies/ million cells
day 11 after UVC I got 20 colonise/million cells
day 16 after UVC I got 10 colonies/million cells

This is just my first time results, I will try to repeat them

-------------------
Question??

Does anyone know if this mutation (HPRT-) frequence will be decreased after some certain of time?

Such as on day 16 after UVC, the number of colonies are decreased from day 11 like 50% or it was my error technique during counting cell or pipetting that made number of colonies drop like that.

//Tai

Hi,

I am starting to use hprt mutation assay and am therefore going through various protocols. One thing I am not sure of and am hoping that some of you might be able to clarify this to me. The S9 treatment, is it necessary to make additional sample with S9 treatment (for metabolic activation)? I will be using ENU (Ethyl-nitrosourea), which does not need metabolic activation, in my assay and am wondering that do I need to make additional sample with metabolic activation? Frankly, I am a bit puzzled about this whole metabolic activation thing... Please, could somebody help me?

Cheers,

J

Hi,

 

Does anyone have a protocol for HPRT assay using human peripheral blood lymphocytes?

 

Cases described above use adherent cells;T lymphocytes grow in suspension. I understand that adherent cells form isolated countable colonies. How do we count colonies with unadherent cells?

 

Also, I am interested in measuring native mutation frequency of patient derived samples and not in MF as a function of mutagen treatment.   

 

Thanks in advance.

SBK

You can't easily do colony forming assays on suspension cells without embedding in agar or something similar (if the cells will tolerate it). 

SBK on Tue Dec 10 06:23:40 2013 said:

Hi,

 

Does anyone have a protocol for HPRT assay using human peripheral blood lymphocytes?

 

Cases described above use adherent cells;T lymphocytes grow in suspension. I understand that adherent cells form isolated countable colonies. How do we count colonies with unadherent cells?

 

Also, I am interested in measuring native mutation frequency of patient derived samples and not in MF as a function of mutagen treatment.   

 

Thanks in advance.

SBK

 

Hi,

This is quite old tread but I will answer if it could help to someone

 

my friend use TFT to select mutation for thymidine kinase on TK6 cells. TK6 is suspension cells and she cast the cells in the low-melting agarose with containing cell medium (she made 2x concentration). Mix 1:1 agarose to 2x medium. It is the same idea of HPRT mutation.