Polly and Molly (born 1997), two ewes, were the first

mammal Mammals () are a group of vertebrate animals constituting the class Mammalia (), characterized by the presence of mammary glands which in females produce milk for feeding (nursing) their young, a neocortex (a region of the brain), fur ...

s to have been successfully

cloned Cloning is the process of producing individual organisms with identical or virtually identical DNA, either by natural or artificial means. In nature, some organisms produce clones through asexual reproduction. In the field of biotechnology, ...

from an adult somatic

cell Cell most often refers to: * Cell (biology), the functional basic unit of life Cell may also refer to: Locations * Monastic cell, a small room, hut, or cave in which a religious recluse lives, alternatively the small precursor of a monastery ...

and to be

transgenic A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques, from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the ...

animals at the same time. This is not to be confused with Dolly the Sheep, the first animal to be successfully cloned from an adult somatic cell where there wasn’t modification carried out on the adult donor nucleus. Polly and Molly, like Dolly the Sheep, were cloned at the

Roslin Institute The Roslin Institute is an animal sciences research institute at Easter Bush, Midlothian, Scotland, part of the University of Edinburgh, and is funded by the Biotechnology and Biological Sciences Research Council. It is best known for creati ...

Edinburgh Edinburgh ( ; gd, Dùn Èideann ) is the capital city of Scotland and one of its 32 Council areas of Scotland, council areas. Historically part of the county of Midlothian (interchangeably Edinburghshire before 1921), it is located in Lothian ...

Scotland Scotland (, ) is a country that is part of the United Kingdom. Covering the northern third of the island of Great Britain, mainland Scotland has a border with England to the southeast and is otherwise surrounded by the Atlantic Ocean to ...

. 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 hopefully be used to produce pharmacological and therapeutic proteins to treat human diseases. The protein in question was the human blood clotting

factor IX Factor IX (or Christmas factor) () is one of the serine proteases of the coagulation system; it belongs to peptidase family S1. Deficiency of this protein causes haemophilia B. It was discovered in 1952 after a young boy named Stephen Christmas ...

. Another difference from Dolly the Sheep was the source

cell type A cell type is a classification used to identify cells that share morphological or phenotypical features. A multicellular organism may contain cells of a number of widely differing and specialized cell types, such as muscle cells and skin cell ...

of the nucleus that was transferred. Although Polly and Molly were nuclear clones, they had different

mtDNA Mitochondrial DNA (mtDNA or mDNA) is the DNA located in mitochondria, cellular organelles within eukaryotic cells that convert chemical energy from food into a form that cells can use, such as adenosine triphosphate (ATP). Mitochondrial DNA ...

that was different from the nuclear cells where they received their DNA. Prior to the production of Polly and Molly, the only demonstrated way to make a transgenic animal was by microinjection of DNA into the

pronuclei A pronucleus () is the nucleus of a sperm or egg cell during the process of fertilization. The sperm cell becomes a pronucleus after the sperm enters the ovum, but before the genetic material of the sperm and egg fuse. Contrary to the sperm cell, ...

of fertilized

oocyte An oocyte (, ), oöcyte, or ovocyte is a female gametocyte or germ cell involved in reproduction. In other words, it is an immature ovum, or egg cell. An oocyte is produced in a female fetus in the ovary during female gametogenesis. The female ...

s (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 cell Embryonic stem cells (ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre- implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist ...

s provide a means to transfer new DNA into the

germline In biology and genetics, the germline is the population of a multicellular organism's cells that pass on their genetic material to the progeny (offspring). In other words, they are the cells that form the egg, sperm and the fertilised egg. They ...

. They also allow precise genetic modifications by

gene targeting Gene targeting (also, replacement strategy based on homologous recombination) is a genetic technique that uses homologous recombination to modify an endogenous gene. The method can be used to delete a gene, remove exons, add a gene and modify ...

. Modified embryonic stem cells can be selected

in vitro ''In vitro'' (meaning in glass, or ''in the glass'') studies are performed with microorganisms, cells, or biological molecules outside their normal biological context. Colloquially called " test-tube experiments", these studies in biology a ...

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 A somatic cell (from Ancient Greek σῶμα ''sôma'', meaning "body"), or vegetal cell, is any biological cell forming the body of a multicellular organism other than a gamete, germ cell, gametocyte or undifferentiated stem cell. Such cells com ...

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, three 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

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, also spelled hemophilia B, is a blood clotting disorder causing easy bruising and bleeding due to an inherited mutation of the gene for factor IX, and resulting in a deficiency of factor IX. It is less common than factor VIII defi ...

of which treatment requires intravenous infusion of factor IX. The production of this protein in livestock milk, a process known as

pharming Pharming is a cyberattack intended to redirect a website's traffic to another, fake site by installing a malicious program on the computer. Pharming can be conducted either by changing the hosts file on a victim's computer or by exploitation of a ...

, 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).

References

{{Reflist 1997 animal births Cloning Cloned sheep Vertebrate developmental biology Cell biology Stem cells Biotechnology Individual animals in the United Kingdom