Polly and Molly

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Polly and Molly (born 1997), two ewes, were the first mammals to have been successfully cloned from an adult somatic cell and to be transgenic animals at the same time. [1] 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 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 hopefully be used to produce pharmacological and therapeutic proteins to treat human diseases. The protein in question was the human blood clotting factor IX. [2] Another difference from Dolly the Sheep was the source cell type of the nucleus that was transferred. Although Polly and Molly were nuclear clones, they had different mtDNA that was different from the nuclear cells where they received their DNA. [3]

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 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, 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". [4] 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 IX 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).

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<span class="mw-page-title-main">Dolly (sheep)</span> First cloned mammal (1996–2003)

Dolly was a female Finn-Dorset sheep and the first mammal that was cloned from an adult somatic cell. She was cloned by associates of the Roslin Institute in Scotland, using the process of nuclear transfer from a cell taken from a mammary gland. Her cloning proved that a cloned organism could be produced from a mature cell from a specific body part. Contrary to popular belief, she was not the first animal to be cloned.

<span class="mw-page-title-main">Somatic cell nuclear transfer</span> Method of creating a cloned embryo by replacing the egg nucleus with a body cell nucleus

In genetics and developmental biology, somatic cell nuclear transfer (SCNT) is a laboratory strategy for creating a viable embryo from a body cell and an egg cell. The technique consists of taking an denucleated oocyte and implanting a donor nucleus from a somatic (body) cell. It is used in both therapeutic and reproductive cloning. In 1996, Dolly the sheep became famous for being the first successful case of the reproductive cloning of a mammal. In January 2018, a team of scientists in Shanghai announced the successful cloning of two female crab-eating macaques from foetal nuclei.

In cellular biology, a somatic cell, 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 compose the body of an organism and divide through the process of binary fission and mitotic division.

Sir Ian Wilmut, OBE FRS FMedSci FRSE is an English embryologist and Chair of the Scottish Centre for Regenerative Medicine at the University of Edinburgh. He is best known as the leader of the research group that in 1996 first cloned a mammal from an adult somatic cell, a Finnish Dorset lamb named Dolly. He was appointed OBE in 1999 for services to embryo development and knighted in the 2008 New Year Honours. He together with Keith Campbell and Shinya Yamanaka jointly received the 2008 Shaw Prize for Medicine and Life Sciences "for their works on the cell differentiation in mammals."

<span class="mw-page-title-main">Nuclear transfer</span> Form of cloning

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A biologist is a scientist who conducts research in biology. Biologists are interested in studying life on Earth, whether it is an individual cell, a multicellular organism, or a community of interacting populations. They usually specialize in a particular branch of biology and have a specific research focus.

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<span class="mw-page-title-main">Microinjection</span>

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<span class="mw-page-title-main">Biotechnology in pharmaceutical manufacturing</span>

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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 transgenic animal. Megan and Morag, like Dolly and Polly, were cloned at the Roslin Institute in Edinburgh, Scotland in 1995.

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<span class="mw-page-title-main">Keith Campbell (biologist)</span> British biologist (1954–2012) Professor of Animal Development at the University of Nottingham,

Keith Henry Stockman Campbell was a British biologist who was a member of the team at Roslin Institute that in 1996 first cloned a mammal, a Finnish Dorset lamb named Dolly, from fully differentiated adult mammary cells. He was Professor of Animal Development at the University of Nottingham. In 2008, he received the Shaw Prize for Medicine and Life Sciences jointly with Ian Wilmut and Shinya Yamanaka for "their works on the cell differentiation in mammals".

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<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

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<i>In re Roslin Institute</i> (Edinburgh) 2014 decision of the United States Court of Appeals for the Federal Circuit

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Grahame Bulfield, CBE, FRSE, Hon FRASE is an English geneticist, vice-principal and Emeritus Professor of Genetics at the University of Edinburgh. He is best known as the former director and chief executive of the Roslin Institute, Edinburgh, when in 1996 the research group led by Ian Wilmut first cloned a mammal from an adult somatic cell, a Finnish Dorset lamb named Dolly.

References

  1. McCreath, K. J.; Howcroft, J.; Campbell, K. H. S.; Colman, A.; Schnieke, A. E.; Kind, A. J. (29 June 2000). "Production of gene-targeted sheep by nuclear transfer from cultured somatic cells". Nature. 405 (6790): 1066–1069. doi:10.1038/35016604. PMID   10890449. S2CID   4419356.
  2. AE Schnieke; et al. (1997). "Human factor IX transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts". Science. 278 (5346): 2130–2133. doi:10.1126/science.278.5346.2130. PMID   9405350.
  3. Evans, Matthew J.; Gurer, Cagan; Loike, John D.; Wilmut, Ian; Schnieke, Angelika E.; Schon, Eric A. (September 1999). "Mitochondrial DNA genotypes in nuclear transfer-derived cloned sheep". Nature Genetics. 23 (1): 90–93. doi:10.1038/12696. ISSN   1546-1718. PMC   3042135 . PMID   10471506.
  4. Alan Colman (November 1999). "Dolly, Polly and other 'ollys': likely impact of cloning technology on biomedical uses of livestock". Genet Anal. Genetic Analysis: Biomolecular Engineering. 15 (3–5): 167–173. doi:10.1016/s1050-3862(99)00022-4. PMID   10596758.