[-Saturday, January 20, 2007-]

Wad hab i learnt tis week?

I realised that my previous post is filled with "la", "wad", "wtf".. LoLx!! Read liao feel so weird.

Anyway, this week quite interesting. Get to learn more abt cancer and transgenic zebra fish.

Fri got biotech's practical where we got to inject promoter-specific DNA construct which will be able to make use of zebra fish's protein synthesis mechanism to produce Green/Red fluorescene protein. A typical commercialised fish on sale is called Glo-Fish.

We got to play with Skin-specific and muscle specfic promoter DNA which the fluorscene protein will be produced on the skin and muscle respectively.

First, we caught 2 fishes and put them into a tank of phenylethanol where they will be anaesthesized. They will turned upside down and stopped moving after 30-60 seconds. Next, we will take them out and inject 1 micro litre of DNA construct into the muscles below their fin. After that, we transferred them into a tank filled with antibiotics to prevent them suffering from inflammation from the injuries. Finally, the fishes are transferred to a clean fish tank of normal water. Each of us injected 5 fishes each.

After that, the teaching assistant (TA) proceeded to bring to observe the Glo-Fish on the other side. According to him, the Glo-Fish are stable trangenic line which means that the DNA construct which we had injected are present in their chromosomes and will stay with them through life. They will be able to produce the fluorescene for the whole lifespan and their babies will most likely able to inherit these traits as well. For the fishes we had injected, they will only be able to stay fluorescene for half a year as they are of a transcient expression. That is the DNA will be lost gradually overtime as the DNA are injected intramuscularly.

If we want the DNA to stuck with the animal, we hab to inject the DNA into the embryos of these fishes. This will increase the chance of allowing the fishes to retain these DNA within their genome. In order to inject into these small embryos, we hab to use micro injection which is the last part of the practical. However, the success rate of injecting, alllowing the embryos to survive till they become fishes and the fishes eventually have the DNA stuck with them are still comparably low. For those embryos which hab survived to become fishes, some will fluorscene while some will not. Those which are able to fluorscence, we mate them with the normal zebra fishes to give a F1 generation.

Those in F1 generation which possess this phenotype (traits of able to fluorscence, less than 5%), we called them to be the transgenic founder because they contained the DNA within them but we cannot be sure whether this traits will stay with them for life. For some which do not fluorscence, this does not mean that they do not possess the DNA within them, there is a small probability that they do possess the DNA however, they are called silent gene (unable to be expressed phenotypically but they are within the animal chromosomes genotypically).

Eg. For human. Guy germline DNA consists of XY whereas girl consists of XX. As girl's DNA consist of 2 same X and X. One of the X are being inactivated and knowned as the Barr Bodies. This is one of the reasons why there are alot of genetic dieseases which there's a high possiblity of woman suffering from. As only one of the X DNA is expressed in female to allow proper functioning of the cells, hence if the defective X DNA chromatid is transmitted from the mother or the father (most likely from the mother side who is the carrier of the genetic disease) to a girl. Her body will only b able to express this defective X DNA template for the functioning of cells if the other normal X DNA is being inactivated. The inactivation process is random and will change randomly during each cell division. Normally, this defective X DNA causes loss of function. For eg. The normal X DNA consists of gene which allow expression of proteins to form your immune system but this defective X DNA gene which codes for the same function is changed to a non-functional proteins. Therefore, your imune system is ruined. However, there are some gene being mutated that gives a gain of function gene. However, the proteins being expressed by such gene may be good or bad for you. It may boost your immune system compared to the normal gene or it may act as a poison to your body.

Back to the zebra fish, since there is such process where in case of a double XX chromosomes where 1 of the X will be inactivated. This X may be the chromosome that possess our fluorscence DNA but it was not able to be expressed as it was being inactivated. Therefore, in order to screen for such possibility, we uses RT-PCR (Reverse time Poly Chain Reaction) where the DNA are being amplified and analysed by SDSpage/ Electrophoresis/ELISA methods. If the DNA sequence of the DNA which we had injected earlier are found, this showed that this fish is also a trangenic founder.

So what's the difference between a transgenic founder and a stable transgenic line?
Transgenic founder is the F1 generation. Whereas a stble transgenic line is the F2 generation. F2 generation is when the transgenic founders are mated with the normal zebra fishes again. The % of the F2 generation having the fluorscence trait is 50% according to the Mendelian Rule. For the transgenic founder, theres a possibilty the DNA which we hab injected via their parents be lost. However, for the stable transgenic line, the DNA hab been integrated inside their own genome. Loss of the trangenic DNA is of low porbability.

What are the consequences of such transgenic fish if they were to be released into the wild?
Will they cause the wild type species of zebra fish to be extinct? Previously, theres 2 mathematicians who proposed that such Glo-Fish will be more popular to the female wild type zebra fishes since they FLUORSCENCE and APPEAL to US. No experimental studies were conducted to confirm this. Finally, experiment were conducted and the results were totally opposite of the 2 mathematicians.

First, Male zebra fishes that FLUORSCENCE DO NOT APPEAL to both the Female wild type and transgenic zebra fishes. They only appeal to us =="

Imagine Hulk and Superman come to you and want you to choose one of them. Of cos, you will choose Superman right? LoLx!! Okay, my analogy abit off. =.=

Second, transgenic zebra fishes lifespan is shorter.

Conclusion?? U figure that out urself.

Next, we went to do the micro injection. The needle is super fine.. I think poke into u u oso duno lo. So we use a microscope and proceed to aim the needle towards the embryos. The embryos like soccer ball lo. The first time i poked, i poked through it =.= Den u can see the embryos from round became deflated. Dots.. I killed 1 fish =X Den i tried again. This time succeeded. Woot!! Hope they will grow wen we come back on Monday. ^^
"Never ask why i love you, just accept that i do." || 6:59 PM