One of the simplest cloning techniques works very well on fish species. The technique is called parthenogenesis. It's reproduction without fertilization by male gamete. The eggs develop without sperm and are clones of the female parent. A group of us toyed with this technique back in college.
We took mature ornate bichir females and stripped them of their eggs. We then manually inserted nuclei from the adults red blood cells to provide the full set of paired chromosomes required for egg development.
After many trials and failures, here's a method that produced several developing eggs and subsequent fry:
First, fresh blood samples were taken from adult ornates. Fish have nucleated blood cells as opposed to non-nucleated mammal blood cell (new cells formed n marrow). The blood was placed in petri dishes containing nutrient agar. A heated and stretched out pipette is used to extract the nucleus from within the blood cell. We had to employ rubber sacs filled with mercury to give us micrometer-like movement within the modified pipette syringe to be able to extract the nuclei without much hemoglobin material. The syringe was set to withdraw or inject by way of a lamp turned on and off to expand/contract the mercury sac. Once the syringe was charged it was time for egg preparation. The nucleus within the egg had to be destroyed since the full set of chromosomes would be provided by the blood cell nucleus. We used a short-wave light source to damage the eggs nucleus. Once the nucleus is destroyed, the egg is only viable for a max of 10 minutes before the rest of the cell dies. The light-treated egg is manuevered, in its' petri dish, to the base holding the syringe. A dissecting microscope base micrometer is used to manipulate egg/petri dish until the syringe tip penetrates the egg membrane. Once inside the egg cell, the lamp is lit to expand the mercury sac and inject the nucleus into the egg. The penetration of the egg membrane also cause a hormone to seek out the male gamete to signal the egg that it has been fertilized. The chromosomes are within the injected nucleus and the egg will begin differentiating (if the short-wave light didn't damage any other portions of the cell).
It took 4 of us, working in tandem, to get only 15 of 200 eggs to develop. All of the developing fry were, of course, female since that was the source of the genetic material.
The procedure is viable as a small-scale method. And, it's cost is minimal. The dissection microscope was "borrowed" from the lab. The other materials came to less than $100 back in '75 when we attempted this procedure.
We took mature ornate bichir females and stripped them of their eggs. We then manually inserted nuclei from the adults red blood cells to provide the full set of paired chromosomes required for egg development.
After many trials and failures, here's a method that produced several developing eggs and subsequent fry:
First, fresh blood samples were taken from adult ornates. Fish have nucleated blood cells as opposed to non-nucleated mammal blood cell (new cells formed n marrow). The blood was placed in petri dishes containing nutrient agar. A heated and stretched out pipette is used to extract the nucleus from within the blood cell. We had to employ rubber sacs filled with mercury to give us micrometer-like movement within the modified pipette syringe to be able to extract the nuclei without much hemoglobin material. The syringe was set to withdraw or inject by way of a lamp turned on and off to expand/contract the mercury sac. Once the syringe was charged it was time for egg preparation. The nucleus within the egg had to be destroyed since the full set of chromosomes would be provided by the blood cell nucleus. We used a short-wave light source to damage the eggs nucleus. Once the nucleus is destroyed, the egg is only viable for a max of 10 minutes before the rest of the cell dies. The light-treated egg is manuevered, in its' petri dish, to the base holding the syringe. A dissecting microscope base micrometer is used to manipulate egg/petri dish until the syringe tip penetrates the egg membrane. Once inside the egg cell, the lamp is lit to expand the mercury sac and inject the nucleus into the egg. The penetration of the egg membrane also cause a hormone to seek out the male gamete to signal the egg that it has been fertilized. The chromosomes are within the injected nucleus and the egg will begin differentiating (if the short-wave light didn't damage any other portions of the cell).
It took 4 of us, working in tandem, to get only 15 of 200 eggs to develop. All of the developing fry were, of course, female since that was the source of the genetic material.
The procedure is viable as a small-scale method. And, it's cost is minimal. The dissection microscope was "borrowed" from the lab. The other materials came to less than $100 back in '75 when we attempted this procedure.
When I was in college I drank Heineken and took LSD. Maybe I should have taken bio.
