Polly and Molly
Encyclopedia
Polly and Molly two ewes
, were the first mammal
s to have been successfully cloned
from an adult somatic cell
and to be transgenic animals at the same time. This is not to be confused with Dolly the Sheep
which was the first animal to be successfully cloned from an adult somatic cell where there was no genetic modification carried out on the adult donor nucleus. Polly and Molly, like Dolly the Sheep, were cloned at the Roslin Institute in Edinburgh
, Scotland
.
The creation of Polly and Molly built on the somatic nuclear transfer experiments that led to the cloning of Dolly the Sheep
. The crucial difference was that in creating Polly and Molly, scientists used cells into which a new gene had been inserted. The gene chosen was a therapeutic protein to demonstrate the potential of such recombinant DNA technology combined with animal cloning. This could produce pharmacological and therapeutic proteins to hopefully treat human diseases. The protein in question was the human blood clotting factor IX. Another difference from Dolly the Sheep was the source cell type of the nucleus that was transferred. In the case of Dolly the Sheep, the nucleus that was transferred came from mammary gland cells from a 6-year-old ewe; in the case of Polly and Molly, fibroblast cells were used.
of DNA into the pronuclei of fertilized oocytes (eggs). However, only a small proportion of the animals will integrate the injected DNA into their genome. In the rare cases that they do integrate this new genetic information, the pattern of expression of the injected transgene's protein due to the random integration is very variable. As the aim of such research is to produce an animal that expresses a particular protein in high levels in, for example, its milk, Microinjection
is a very costly procedure that does not usually produce the desired animal.
In mice, there is an additional option for genetic transfer that is not available in other animals. Embryonic stem cells provide a means to transfer new DNA into the germline
. They also allow precise genetic modifications by gene targeting
. Modified embryonic stem cells can be selected in vitro before the experiment moves on further for the production of an animal. Embryonic stem cells capable of contributing to the germline
of livestock species such as sheep have not been isolated.
The production of Dolly the Sheep
and also Megan and Morag
, the two sheep that led to the production of Dolly, demonstrated that viable sheep can be produced by nuclear transfer
from a variety of somatic cell types which have been cultured in vitro. Polly and Molly represented the further step in which somatic cells were cultured in vitro, just as in the case with the previous sheep. However, in this case they were transfected with foreign DNA, and the transfected cells which stably integrated this new piece of genetic information were selected. The nuclei of these somatic cells was then transferred into an empty oocyte, as in the procedure of nuclear transfer
and this was used to produce several transgenic animals. A cell type PDFF was used. PDFF5 would produce male animals and were not transduced. Cell type PDFF2 produced female animals and were transduced. Of the gestations that occurred, 3 PDFF2 animals were born, two of which survived birth 7LL8 and 7LL12. These animals were transfected but contained a marker gene not the cloned gene of interest. These were named "Holly" and "Olly". Two more subsets of female producing PDFF2 cells, PDFF2-12 and PDFF2-13, also produced animals which had the cell of interest together with the marker. Of these lambs 7LL12, 7LL15, and 7LL13 were born alive and healthy. Two of these were named Polly and Molly.
protein in the milk of sheep. This protein plays an essential role in blood coagulation, and deficiency leads to the disease Haemophilia B
of which treatment requires intravenous infusion of factor IX
. The production of this protein in livestock milk, a process known as pharming
, would provide a source of this therapeutic protein that would reduce the cost and also would be free of potential infectious risk associated with the current source of this protein (human blood).
Domestic sheep
Sheep are quadrupedal, ruminant mammals typically kept as livestock. Like all ruminants, sheep are members of the order Artiodactyla, the even-toed ungulates. Although the name "sheep" applies to many species in the genus Ovis, in everyday usage it almost always refers to Ovis aries...
, were the first mammal
Mammal
Mammals are members of a class of air-breathing vertebrate animals characterised by the possession of endothermy, hair, three middle ear bones, and mammary glands functional in mothers with young...
s to have been successfully cloned
Cloning
Cloning in biology is the process of producing similar populations of genetically identical individuals that occurs in nature when organisms such as bacteria, insects or plants reproduce asexually. Cloning in biotechnology refers to processes used to create copies of DNA fragments , cells , or...
from an adult somatic cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
and to be transgenic animals at the same time. This is not to be confused with Dolly the Sheep
Dolly the Sheep
Dolly was a female domestic sheep, and the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer. She was cloned by Ian Wilmut, Keith Campbell and colleagues at the Roslin Institute near Edinburgh in Scotland...
which was the first animal to be successfully cloned from an adult somatic cell where there was no genetic modification carried out on the adult donor nucleus. Polly and Molly, like Dolly the Sheep, were cloned at the Roslin Institute in Edinburgh
Edinburgh
Edinburgh is the capital city of Scotland, the second largest city in Scotland, and the eighth most populous in the United Kingdom. The City of Edinburgh Council governs one of Scotland's 32 local government council areas. The council area includes urban Edinburgh and a rural area...
, Scotland
Scotland
Scotland is a country that is part of the United Kingdom. Occupying the northern third of the island of Great Britain, it shares a border with England to the south and is bounded by the North Sea to the east, the Atlantic Ocean to the north and west, and the North Channel and Irish Sea to the...
.
The creation of Polly and Molly built on the somatic nuclear transfer experiments that led to the cloning of Dolly the Sheep
Dolly the Sheep
Dolly was a female domestic sheep, and the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer. She was cloned by Ian Wilmut, Keith Campbell and colleagues at the Roslin Institute near Edinburgh in Scotland...
. The crucial difference was that in creating Polly and Molly, scientists used cells into which a new gene had been inserted. The gene chosen was a therapeutic protein to demonstrate the potential of such recombinant DNA technology combined with animal cloning. This could produce pharmacological and therapeutic proteins to hopefully treat human diseases. The protein in question was the human blood clotting factor IX. Another difference from Dolly the Sheep was the source cell type of the nucleus that was transferred. In the case of Dolly the Sheep, the nucleus that was transferred came from mammary gland cells from a 6-year-old ewe; in the case of Polly and Molly, fibroblast cells were used.
Background
Prior to the production of Polly and Molly, the only demonstrated way to make a transgenic animal was by MicroinjectionMicroinjection
Microinjection refers to the process of using a glass micropipette to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the...
of DNA into the pronuclei of fertilized oocytes (eggs). However, only a small proportion of the animals will integrate the injected DNA into their genome. In the rare cases that they do integrate this new genetic information, the pattern of expression of the injected transgene's protein due to the random integration is very variable. As the aim of such research is to produce an animal that expresses a particular protein in high levels in, for example, its milk, Microinjection
Microinjection
Microinjection refers to the process of using a glass micropipette to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the...
is a very costly procedure that does not usually produce the desired animal.
In mice, there is an additional option for genetic transfer that is not available in other animals. Embryonic stem cells provide a means to transfer new DNA into the germline
Germline
In biology and genetics, the germline of a mature or developing individual is the line of germ cells that have genetic material that may be passed to a child.For example, gametes such as the sperm or the egg, are part of the germline...
. They also allow precise genetic modifications by gene targeting
Gene targeting
Gene targeting is a genetic technique that uses homologous recombination to change an endogenous gene. The method can be used to delete a gene, remove exons, add a gene, and introduce point mutations. Gene targeting can be permanent or conditional...
. Modified embryonic stem cells can be selected in vitro before the experiment moves on further for the production of an animal. Embryonic stem cells capable of contributing to the germline
Germline
In biology and genetics, the germline of a mature or developing individual is the line of germ cells that have genetic material that may be passed to a child.For example, gametes such as the sperm or the egg, are part of the germline...
of livestock species such as sheep have not been isolated.
The production of Dolly the Sheep
Dolly the Sheep
Dolly was a female domestic sheep, and the first mammal to be cloned from an adult somatic cell, using the process of nuclear transfer. She was cloned by Ian Wilmut, Keith Campbell and colleagues at the Roslin Institute near Edinburgh in Scotland...
and also Megan and Morag
Megan and Morag (cloned sheep)
Megan and Morag, two domestic sheep, were the first mammals to have been successfully cloned from differentiated cells. They are not to be confused with Dolly the Sheep which was the first animal to be successfully cloned from an adult somatic cell or Polly the sheep which was the first cloned and...
, the two sheep that led to the production of Dolly, demonstrated that viable sheep can be produced by nuclear transfer
Nuclear transfer
Nuclear Transfer is a form of cloning. The steps involve removing the DNA from an oocyte , and injecting the nucleus which contains the DNA to be cloned. In rare instances, the newly constructed cell will divide normally, replicating the new DNA while remaining in a pluripotent state...
from a variety of somatic cell types which have been cultured in vitro. Polly and Molly represented the further step in which somatic cells were cultured in vitro, just as in the case with the previous sheep. However, in this case they were transfected with foreign DNA, and the transfected cells which stably integrated this new piece of genetic information were selected. The nuclei of these somatic cells was then transferred into an empty oocyte, as in the procedure of nuclear transfer
Nuclear transfer
Nuclear Transfer is a form of cloning. The steps involve removing the DNA from an oocyte , and injecting the nucleus which contains the DNA to be cloned. In rare instances, the newly constructed cell will divide normally, replicating the new DNA while remaining in a pluripotent state...
and this was used to produce several transgenic animals. A cell type PDFF was used. PDFF5 would produce male animals and were not transduced. Cell type PDFF2 produced female animals and were transduced. Of the gestations that occurred, 3 PDFF2 animals were born, two of which survived birth 7LL8 and 7LL12. These animals were transfected but contained a marker gene not the cloned gene of interest. These were named "Holly" and "Olly". Two more subsets of female producing PDFF2 cells, PDFF2-12 and PDFF2-13, also produced animals which had the cell of interest together with the marker. Of these lambs 7LL12, 7LL15, and 7LL13 were born alive and healthy. Two of these were named Polly and Molly.
Transgene
The transgene that was inserted in the donor somatic cells was designed to express the human clotting factor IXFactor IX
Factor IX is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes hemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas was found to be lacking this exact factor, leading to...
protein in the milk of sheep. This protein plays an essential role in blood coagulation, and deficiency leads to the disease Haemophilia B
Haemophilia B
Haemophilia B is a blood clotting disorder caused by a mutation of the Factor IX gene, leading to a deficiency of Factor IX. It is the second most common form of haemophilia, rarer than haemophilia A. It is sometimes called Christmas disease after Stephen Christmas, the first patient described...
of which treatment requires intravenous infusion of factor IX
Factor IX
Factor IX is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes hemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas was found to be lacking this exact factor, leading to...
. The production of this protein in livestock milk, a process known as pharming
Pharming (genetics)
Pharming is a portmanteau of farming and "pharmaceutical" and refers to the use of genetic engineering to insert genes that code for useful pharmaceuticals into host animals or plants that would otherwise not express those genes. As a consequence, the host animals or plants then make the...
, would provide a source of this therapeutic protein that would reduce the cost and also would be free of potential infectious risk associated with the current source of this protein (human blood).