Can I clone or construct whole Mito DNA to vector

2 posts / 0 new
Last post
Robertwe
Robertwe's picture
Can I clone or construct whole Mito DNA to vector

Can I clone or construct whole mito DNA to vector, then transfect into cells to correct mitochondrial disorder?

RLS
RLS's picture
1. You have to consider

1. You have to consider whether or not the mitochondrial disorder is caused by a problem with a gene within the mitochondria or by a protein or RNA that is imported into the mitochondria.

2. You can make a cDNA library from the mRNA within the mitochondria; first you would need to isolate mitochondria, treat the exterior of the mitochondria with micrococcal nuclease, and extract the mRNA. This requires the polyA tail found at the 3'-ends of the mRNAs. You use reverse transcriptase to reverse transcribe the RNA into DNA. There are a number of kits out there to help you do this. 

3. This will not account for any of the noncoding RNAs in mitochondria such as tRNAs, rRNAs, etc. What if your defect is caused by one of these?

4. You can create a genomic library. Again, you would need to purify mitochondria and extract the genomic DNA. These are more difficult to make and maintain. Basically, you are cloning unique fragments of genomic DNA into a vector. The pool of DNA + vectors is then propagated in bacteria or yeast so that each organism contains one construct (1 vector + 1 piece of DNA). Then the question is how do you get the the products of your library into mitochondria? Mitochondrial transformation is quite difficult. 

What organism are you working with?