How do you make alive mutants of an essential process?

[sci-ki]

Can you please give me some suggestions and basic guidelines on the topic.

 

Thanks

[insane_alien]

can you be more specific and understandable?

[Yggdrasil]

It depends on many factors, but there are various strategies for making lines with lethal mutations. If you are developing a mutant for use in experiments, you can make the knockout tissue specific, for example by using recombinase sites where the recombinase is expressed in a tissue specific manner. For others, you can use a temperature-sensitive mutant, where the protein is active at low temperature but becomes inactivated at a higher temperature. You can also introduce a gene encoding shRNA with an inducible/tissue-specific promoter to knockdown expression of your target gene by RNAi.

[sci-ki]

Thanks a lot...I am mainly interested in the temperature-sensitive mutants. Can you explain the method in more details. Thanks again!

[zyncod]

Well, you mutate a bunch of organisms (w/EMS, UV, etc.), and grow them up at room temp. Usually, temp-sens mutants are single-celled organisms, so you replate them and grow them at 37C. Whatever dies at that temperature harbors a temp-sens mutation.

[Immunologist]

Ok, first answer:

A temperature-sensitive mutant is a mutant bearing a change in amino acid (in a specific protein). This change result in the instability of a protein at high temperature.

For example, if you remove an amino acid that was allowing a solid intra-protein bound, at high temperature, the protein will be denatured because of this missing link.

 

Second, you can also create "conditional mutants". They are mutant where you need to add a reagent (an antibiotic for example) so that a gene is expressed.

 

Hope it helps.

[Peter Gariae]

It depends on many factors, but there are various strategies for making lines with lethal mutations. If you are developing a mutant for use in experiments, you can make the knockout tissue specific, for example by using recombinase sites where the recombinase is expressed in a tissue specific manner. For others, you can use a temperature-sensitive mutant, where the protein is active at low temperature but becomes inactivated at a higher temperature. You can also introduce a gene encoding shRNA with an inducible/tissue-specific promoter to knockdown expression of your target gene by RNAi.

 

More important to understand the contradiction between weak hydrogen bonds base pairs in DNA and codon-anticodon at termophylic bacterium. Some of them are survive at temp. above 100 C. What's about H-bonds?