Question

Glofish is a type of genetically engineered animal. The transgene encodes for an algae flourescent protein which makes the fish glow. describe concentually how a glofish is generated using recombinant DNA technologies. You have to use atleast 3 DNA technologies in your answer.

Answer 1

Algal cells are  subjected to number of enzymes such as cellulase, proteases etc to separate the DNA from rest of the molecules from the cell. Then the genome is subjected to restriction enzyme which cuts the DNA in to many fragments. Restriction enzymes cut DNA at specific sites called recognition sites where they have specific DNA sequences called palindromic sequences producing many DNA fragments.These fragments of the DNA are separated in to bands by using gel-electrophoresis.

Gel electrophoesis is a technique where we load the algal genome that is cut with restriction enzyme on an agarose gel and suject it to current. The DNA fragments cut by the restriction enzyme move towards the + electrode as they vibrate with in the gel. During this process, the smaller fragments move farther away towards the +ve electrode than the larger fragments. Based on the size of the fragment DNA gets separated in to bands. The desired gene for flourescence is identified by using appropriate probes and separated from the gel.

The DNA can be subjected to PCR ( polymerase chain reaction). In this technique , by subjecting the DNA to thermal shockes, DNA is replicated under laboratory conditions. Temperature is increased to 90 C to denature the DNA. Again the temperature is rreduced to about 70C to anneal the primer followed by extension of the new strand by the addition of the nucleotides . The same steps follow...Denaturing, Annealing and Extension to give rise to millions of copies of the same gene.

This is the technique to produce multiple copies of the desired DNA.

The gene of interest here , the fluorescent gene, is introduced in to the fish zygote. Microinjection is one technique , by which the DNA can be injected in to animal cells. The microinjection will directly place the fluorescent gene in to the nucleus of the fish. The gene is flanked with transposase enzyme which will allow it attach to the fish genome. This is how the gene is introduced in to the genome of the zygote. The fish that develops from the zygote will produce the protein needed for glow.