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How to replace gene with modified gene [unequal length] ?

Genetics as it applies to evolution, molecular biology, and medical aspects.

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How to replace gene with modified gene [unequal length] ?

Postby Hicsuntdrac0nis » Mon Mar 19, 2012 5:38 am

How could one replace a gene with a modified gene ? I know how homologous recombination works in gene therapy but can one use homologous recombination if the replacement gene was longer than the original gene ? I know one can put the sequence into an expression vector or some type of virus but that would be introducing the gene into a diffeent site while the original gene is still around . Is there any mechanism that would allow this unequal crossing over ?

My idea was to fuse GFP to the end of a particular gene so all of its protein products fluoresce . How would one replace the original gene with the fused genetic information .
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Postby JackBean » Mon Mar 19, 2012 1:23 pm

The length doesn't matter much. You can easily do deletions or insertions with homologous recombination.
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Re:

Postby Hicsuntdrac0nis » Wed Mar 21, 2012 12:11 am

JackBean wrote:The length doesn't matter much. You can easily do deletions or insertions with homologous recombination.


During recombination when:

Original Segment A = T-A-T-C-T-T-T-T-A-G-G-T-A-G-C-A
Engineer Segment B = T-A-T-C-T-T-T-T-C-C-C-C-C-G-C-A

The sequences would switch places, but what if the segment after the original gene [A-G-G-T-A-G-C-A in this case] was important and could not be deleted or modified for other cellular functions . If you fused two sequences together [Segment B] and then put homologous sequence after it so it would recombind, some of the original genomic information [following Segment A] would be lost .

So how would you replace a gene with a modified gene without perturbing any other genomic information ?
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Postby JackBean » Wed Mar 21, 2012 11:23 am

you can always include that information which would be deleted, in your construct. Although that would be somewhat complicated.
From what I remember from introducing pGAPZ and pPICZ vectors into Pichia, they just contain the promotor, which is usually in the yeast genome and the vector is cut in the promotor to be linearized and then simply inserted so that both genes are working.

Maybe do not use particular sequence, just some marks like A for homologous sequence 1, B for sequence in between and C for homologous sequence 2, so that you get A-B-C. Now, what should be the problem?
http://www.biolib.cz/en/main/

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