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BAC for transgenic mice - (May/08/2011 )

Hi, i'm going to start on creating transgenic mice using BAC. My question is, is there a need to sequence the full BAC (almost 200kb)to ensure that everything is correct before i start manipulating the BAC via recombineering? I've tried to PCR out regions of the genes of my interest using the BAC clone as template and i've managed to get specific bands of right size. I'm in the midst of doing end-sequencing, and is planning to do a restriction digest map. However, none of these would ensure that the BAC is absolutely correct unless I sequence.

Do anyone know the standard practice for this? Do I need to ensure every single basepair is correct via sequencing or is whatever that i'm planning to do sufficient to give the green light to start working with the BAC?

Thanks a lot!

-krystle-

I've not used a BAC for creating transgenic mice, but do routinely use a BAC version of our virus for making mutants. We don't sequence the whole BAC every time, it would be prohibitively expensive for our work. We do sequence our region of interest and then do RFLP using several different RE to ensure no major errors/changes have occured. This is the published standard for our field.
Hope that helps? :)

-leelee-

leelee on Mon May 9 08:39:01 2011 said:


I've not used a BAC for creating transgenic mice, but do routinely use a BAC version of our virus for making mutants. We don't sequence the whole BAC every time, it would be prohibitively expensive for our work. We do sequence our region of interest and then do RFLP using several different RE to ensure no major errors/changes have occured. This is the published standard for our field.
Hope that helps? :)



Hi, thanks for your reply. The region of interest we're looking at is 20+kb, so i guess i should at least sequence that. I'm not really familiar with RFLP. I know for RFLP should do restriction enzyme digestion, run through agarose gel, and do a southern blot right? But for southern blot you need a probe, how do you design that probe/how to decide which region to use for the probe? Also for the probe, is it going to be radiolabelled or is there non-radiolabelled form like biotinlyated? Is it as good if I just do a restriction enzyme digestion map, in other words, do the digestion and run through gel via pulse-field (can't do normal gel electrophoresis right?)?

As for purifying the BAC, do you use kit or any particular method? I found the miniprep protocol on bac.chori.org and was wondering if that is sufficient because i did see other protocols online like using CsCl gradient.

Sorry for the many question as i'm really new with handling BAC.

Thanks so much.

-krystle-

For my RFLPs I don't do a southern, I run the digests by standard AGE. I do a restriction map for the BAC with my mutation and then make sure the actual pattern that I get by AGE is correct.

All this said, our parental BAC (from which all mutants are made) has been fully sequenced by our lab after it was constructed.
Did you make the BAC yourself or get it from someone else? Do they have the sequence?
If you constructed the BAC yourself, it may be worth getting it sequenced to begin with.

-leelee-

Our BAC is about 230kb.
If I'm just screening during BAC mutagenesis, I'll do a basic mini prep, but once I'm finished, I'll do up a bigger prep and extract using the midi-kit from Machery-Nagel. I'm pretty sure we use the NucleoBond Xtra Midi kit, but I can get the kit cat number for you if you like?

-leelee-

Hi, thanks so much for your information. The BAC I have is purchased from bac.chori.org. I tried to PCR certain fragment of the region of interest and i was able to get a specific band. So I believe the BAC clone is correct. So i will probably do the restriction digest map to verify that. The bac just contains a genomic region of mouse genome, so the sequence is available on ncbi. But i'm not sure if this means that the bac clone we purchased has the exact same sequence, and i'm not sure what is the standard practice of dealing with bac to verify that. And the website for the organisation also stated that they only generate and keep stock the bac clones, they don't sequence to ensure they are definitely correct.

For the kit, i'll just google myself. So since you use the kit i assume that it has no issue with shearing the bac?

Also, do you know if i need to sequence the bac, either only around 20kb or the full 200+kb, what is the best method? I read that i could do shot gun sequencing whereby the bac is randomly cleaved to shorter length, ta cloned to plasmid, and sequenced using primers specific for the plasmid. I was wondering if you know of better and easier method. The size i'm looking at is too small to consider next generation sequencing right?


Thanks again!

-krystle-

For sequencing regions of my BAC I just send off the PCR product together with the primers to a facility nearby and they do it for me. Not sure what type of seq they do, I'll ask my supervisor.
And no, no issues with shearing of my BAC with that kit.

-leelee-