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Genetic engineering of a mouse: Dr. Frank Ruddle and somatic cell genetics.

Jones D - Yale J Biol Med (2011)

Bottom Line: Dr. Jon W.Ruddle were pioneers in mammalian gene transfer research.Their research resulted in production of the first transgenic animals, which contained foreign DNA that was passed on to offspring.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, Yale School of Medicine, 10 Amistad Street, New Haven, CT 06519, USA. dennis.jones@yale.edu

ABSTRACT
Genetic engineering is the process of modifying an organism's genetic composition by adding foreign genes to produce desired traits or evaluate function. Dr. Jon W. Gordon and Sterling Professor Emeritus at Yale Dr. Frank H. Ruddle were pioneers in mammalian gene transfer research. Their research resulted in production of the first transgenic animals, which contained foreign DNA that was passed on to offspring. Transgenic mice have revolutionized biology, medicine, and biotechnology in the 21st century. In brief, this review revisits their creation of transgenic mice and discusses a few evolving applications of their transgenic technology used in biomedical research.

Show MeSH
Figure 1 (modified from [38]). Generation of the first transgenic mouse. To create transgenic animals (mice shown here), male and female mice were mated. The single-celled embryo was obtained from the pregnant female. DNA was microinjected into the pronucleus of a fertilized ovum. Once injected, surviving embryos were re-implanted into the oviduct of pseudopregnant female recipients. These females gave birth about 3 weeks after implantation. Transgene integration from litters was assessed by tissue analysis. This DNA injection into the pronucleus was the first and still most commonly used technique to make transgenic animals.
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Figure 1: Figure 1 (modified from [38]). Generation of the first transgenic mouse. To create transgenic animals (mice shown here), male and female mice were mated. The single-celled embryo was obtained from the pregnant female. DNA was microinjected into the pronucleus of a fertilized ovum. Once injected, surviving embryos were re-implanted into the oviduct of pseudopregnant female recipients. These females gave birth about 3 weeks after implantation. Transgene integration from litters was assessed by tissue analysis. This DNA injection into the pronucleus was the first and still most commonly used technique to make transgenic animals.

Mentions: To summarize their work, several recombinant plasmids were created and modified for the purpose of tracking specific genes after introduction into mammalian embryos. The first plasmid, named pST6, was a derivative of pBR322, a widely used bacterial plasmid for cloning at the time. The authors inserted a portion of the SV40 virus and the herpes simplex virus thymidine kinase (TK) gene. These sequences would allow for DNA replication and confirmation of gene transfer, respectively. For negative controls, they created variations of the pST6 plasmid, which included reversal of the SV40 sequence and formation of dimerized pST6 plasmids. These were termed pST9 and pST12, respectively. A second type of plasmid, pRH 1.3Mm 1, also was derived from the pBR322 plasmid. It contained a random and interspersed sequence. If DNA integration was mediated by homologous recombination, this sequence would increase integration frequency, as it was shown that repetitive sequences caused a high frequency of recombination and formation of recombinant genomes in yeast [12]. Next, Dr. Ruddle and Dr. Gordon aimed to inject their purified DNA into single-celled embryos to ensure passage to all daughter cells. However, this would be technically challenging because of the small size of the embryos. To solve this problem, very fine microneedles were created from capillary tubes. In addition, holding pipettes were constructed from capillary tubes in order to stabilize the embryo. By capillary action, DNA was collected into the microneedle for injection. Their recombinant plasmids were then “microinjected” into single-celled mouse embryos at the pronuclear stage of development. Surviving eggs were implanted into psuedopregnant females. Offspring were born 3 weeks later, and they were subjected to DNA analysis by Southern blot (Figure 1). Of the 78 mice injected with the original pST6 plasmid, only two of 78 animals were positive for transformation, as determined by Southern blot hybridization to the pST6 probe. Furthermore, only one animal contained the DNA in integrated form as probed with TK, albeit the gene was modified. The other animal appeared to keep portions of the plasmid DNA without integration of the TK gene. Animals that were uninjected, or injected with the other plasmids mentioned, were not positive for DNA transformation. Despite a low success rate, this study served as proof of principle that cloned DNA could be directly inserted into the mouse genome. Furthermore, incomplete inter-species sequence homology between donor and recipient was not an obstacle to prevent transformation, as human TK was expressed in mice, suggesting that it might be possible to transfer any gene between distinct species. Lastly, failure to see any transformed mice with the pRH 1.3Mm 1 plasmid suggested that this phenomenon appeared to be predominantly random and not mediated by homologous recombination. One year later, Dr. Ruddle and Dr. Gordon reported in Science that these transgenic mice could pass the inserted genes to their offspring [11]. Hence, these sequences had become a stable part of the genome.


Genetic engineering of a mouse: Dr. Frank Ruddle and somatic cell genetics.

Jones D - Yale J Biol Med (2011)

Figure 1 (modified from [38]). Generation of the first transgenic mouse. To create transgenic animals (mice shown here), male and female mice were mated. The single-celled embryo was obtained from the pregnant female. DNA was microinjected into the pronucleus of a fertilized ovum. Once injected, surviving embryos were re-implanted into the oviduct of pseudopregnant female recipients. These females gave birth about 3 weeks after implantation. Transgene integration from litters was assessed by tissue analysis. This DNA injection into the pronucleus was the first and still most commonly used technique to make transgenic animals.
© Copyright Policy - open access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3117405&req=5

Figure 1: Figure 1 (modified from [38]). Generation of the first transgenic mouse. To create transgenic animals (mice shown here), male and female mice were mated. The single-celled embryo was obtained from the pregnant female. DNA was microinjected into the pronucleus of a fertilized ovum. Once injected, surviving embryos were re-implanted into the oviduct of pseudopregnant female recipients. These females gave birth about 3 weeks after implantation. Transgene integration from litters was assessed by tissue analysis. This DNA injection into the pronucleus was the first and still most commonly used technique to make transgenic animals.
Mentions: To summarize their work, several recombinant plasmids were created and modified for the purpose of tracking specific genes after introduction into mammalian embryos. The first plasmid, named pST6, was a derivative of pBR322, a widely used bacterial plasmid for cloning at the time. The authors inserted a portion of the SV40 virus and the herpes simplex virus thymidine kinase (TK) gene. These sequences would allow for DNA replication and confirmation of gene transfer, respectively. For negative controls, they created variations of the pST6 plasmid, which included reversal of the SV40 sequence and formation of dimerized pST6 plasmids. These were termed pST9 and pST12, respectively. A second type of plasmid, pRH 1.3Mm 1, also was derived from the pBR322 plasmid. It contained a random and interspersed sequence. If DNA integration was mediated by homologous recombination, this sequence would increase integration frequency, as it was shown that repetitive sequences caused a high frequency of recombination and formation of recombinant genomes in yeast [12]. Next, Dr. Ruddle and Dr. Gordon aimed to inject their purified DNA into single-celled embryos to ensure passage to all daughter cells. However, this would be technically challenging because of the small size of the embryos. To solve this problem, very fine microneedles were created from capillary tubes. In addition, holding pipettes were constructed from capillary tubes in order to stabilize the embryo. By capillary action, DNA was collected into the microneedle for injection. Their recombinant plasmids were then “microinjected” into single-celled mouse embryos at the pronuclear stage of development. Surviving eggs were implanted into psuedopregnant females. Offspring were born 3 weeks later, and they were subjected to DNA analysis by Southern blot (Figure 1). Of the 78 mice injected with the original pST6 plasmid, only two of 78 animals were positive for transformation, as determined by Southern blot hybridization to the pST6 probe. Furthermore, only one animal contained the DNA in integrated form as probed with TK, albeit the gene was modified. The other animal appeared to keep portions of the plasmid DNA without integration of the TK gene. Animals that were uninjected, or injected with the other plasmids mentioned, were not positive for DNA transformation. Despite a low success rate, this study served as proof of principle that cloned DNA could be directly inserted into the mouse genome. Furthermore, incomplete inter-species sequence homology between donor and recipient was not an obstacle to prevent transformation, as human TK was expressed in mice, suggesting that it might be possible to transfer any gene between distinct species. Lastly, failure to see any transformed mice with the pRH 1.3Mm 1 plasmid suggested that this phenomenon appeared to be predominantly random and not mediated by homologous recombination. One year later, Dr. Ruddle and Dr. Gordon reported in Science that these transgenic mice could pass the inserted genes to their offspring [11]. Hence, these sequences had become a stable part of the genome.

Bottom Line: Dr. Jon W.Ruddle were pioneers in mammalian gene transfer research.Their research resulted in production of the first transgenic animals, which contained foreign DNA that was passed on to offspring.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunobiology, Yale School of Medicine, 10 Amistad Street, New Haven, CT 06519, USA. dennis.jones@yale.edu

ABSTRACT
Genetic engineering is the process of modifying an organism's genetic composition by adding foreign genes to produce desired traits or evaluate function. Dr. Jon W. Gordon and Sterling Professor Emeritus at Yale Dr. Frank H. Ruddle were pioneers in mammalian gene transfer research. Their research resulted in production of the first transgenic animals, which contained foreign DNA that was passed on to offspring. Transgenic mice have revolutionized biology, medicine, and biotechnology in the 21st century. In brief, this review revisits their creation of transgenic mice and discusses a few evolving applications of their transgenic technology used in biomedical research.

Show MeSH