Pig Cloning by Microinjection of Fetal Fibroblast Nuclei

Ōnishi, Akira; Iwamoto, Masao; Akita, Tomiji; Mikawa, Satoshi; Takeda, Kumiko; Awata, Takashi; Hanada, Hirohumi; Perry, Anthony C.F.

doi:10.1126/science.289.5482.1188

Cited by 790 publications

(538 citation statements)

References 21 publications

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“…The targeted cell clones were screened by polymerase chain reaction (PCR) and then used as donor cells for nuclear transfer. The nuclear transfer and subsequent embryo transfer were performed as previously described 17. The cloned fetuses were collected at 39 or 72 days of gestation to confirm the targeting events by Southern blotting and so that large populations of cells could be obtained for further nuclear transfer.…”

Section: Methodsmentioning

confidence: 99%

Development of Human‐Like Advanced Coronary Plaques in Low‐Density Lipoprotein Receptor Knockout Pigs and Justification for Statin Treatment Before Formation of Atherosclerotic Plaques

Fuchimoto

Sudo

et al. 2016

JAHA

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BackgroundAlthough clinical trials have proved that statin can be used prophylactically against cardiovascular events, the direct effects of statin on plaque development are not well understood. We generated low‐density lipoprotein receptor knockout (LDLR −/−) pigs to study the effects of early statin administration on development of atherosclerotic plaques, especially advanced plaques.Methods and Results LDLR −/− pigs were generated by targeted deletion of exon 4 of the LDLR gene. Given a standard chow diet, LDLR −/− pigs showed atherosclerotic lesions starting at 6 months of age. When 3‐month‐old LDLR −/− pigs were fed a high‐cholesterol, high‐fat (HCHF) diet for 4 months (HCHF group), human‐like advanced coronary plaques developed. We also fed 3‐month‐old LDLR −/− pigs an HCHF diet with pitavastatin for 4 months (Statin Prophylaxis Group). Although serum cholesterol concentrations did not differ significantly between the 2 groups, intravascular ultrasound revealed 52% reduced plaque volume in statin‐treated pigs. Pathological examination revealed most lesions (87%) in the statin prophylaxis group were early‐stage lesions, versus 45% in the HCHF diet group (P<0.01). Thin‐cap fibroatheroma characterized 40% of the plaques in the HCHF diet group versus 8% in the statin prophylaxis group (P<0.01), intraplaque hemorrhage characterized 11% versus 1% (P<0.01), and calcification characterized 22% versus 1% (P<0.01).ConclusionsResults of our large animal experiment support statin prophylaxis before the occurrence of atherosclerosis. Early statin treatment appears to retard development of coronary artery atherosclerosis and ensure lesion stability. In addition, the LDLR −/− pigs we developed represent a large animal model of human‐like advanced coronary plaque suitable for translational research.

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Section: Methodsmentioning

confidence: 99%

Development of Human‐Like Advanced Coronary Plaques in Low‐Density Lipoprotein Receptor Knockout Pigs and Justification for Statin Treatment Before Formation of Atherosclerotic Plaques

Fuchimoto

Sudo

et al. 2016

JAHA

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“…With the successful cloning of animals by somatic cell nuclear transfer (SCNT), it is now possible to produce transgenic pigs from genetically engineered somatic donor cells. In SCNT, reported for a variety of animal species including the mouse (Wakayama et al 2000; Yanagimachi 1999), the sheep (Wilmut et al 1997), the cow (Cibelli et al 1998;Kato et al 1998;Kubota et al 2000), and the pig (Betthauser et al 2000;Onishi et al 2000;Polejaeva et al 2000), the nucleus from a single differentiated somatic cell is transferred into an enucleated oocyte (unfertilized egg cell), and the reconstructed embryo is subsequently transferred to a surrogate mother. This procedure allows modification of the somatic donor cell in culture by transgene insertion or introduction of loss-offunction gene knockout mutations by homologous recombination.…”

Section: Introductionmentioning

confidence: 99%

Pig transgenesis by Sleeping Beauty DNA transposition

Jakobsen

Kragh

et al. 2010

Transgenic Res

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Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.

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“…In addition, and again in contrast to conventional micromanipulation, piezo permits multiple procedures to be performed with a single needle. Thus, although many oocytes (such as those of humans, cattle, pigs and rabbits) can be injected conventionally, the relative speed and ease of piezo means that it is advantageous even in these species 3,[15][16][17][18][19][20] . Ease of pipette fabrication (beveled tips are not required) and speed also make piezo suitable for the injection of blastocysts with ES cells 11 , and the method additionally allows partial ablation of the inner cell mass (i.e., the removal of potentially competing embryonic cells).…”

Section: Introductionmentioning

confidence: 99%

Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)

Yoshida¹,

Perry²

2007

Nat Protoc

156

135

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The mouse is a genetically tractable model organism widely used to study mammalian development and disease. However, mouse metaphase II (mII) oocytes are exquisitely sensitive and intracytoplasmic sperm injection (ICSI) with conventional pipettes generally kills them. This problem can be solved with piezo-actuated micromanipulation, in which the piezo-electric effect (crystal deformation in response to an externally applied voltage) propels a microinjection needle tip forward in a precise and rapid movement. Piezoactuated micromanipulation enhances the penetration of membranes and matrices, and mouse ICSI is a major application. Here we describe a comprehensive, step-by-step mouse piezo ICSI protocol for non-specialists that can be completed in 2-4 h. The protocol is a basic prelude to multiple applications, including nuclear transfer cloning, spermatid injection, blastocyst injection, mII transgenesis, and streamlining micromanipulation in primates and livestock. Moreover, piezo ICSI can be used to obtain offspring from 'dead' (non-motile) sperm, enabling trivial sperm freezing protocols for mouse strain storage and shipment. INTRODUCTIONPiezo-actuated micromanipulation harnesses the piezo-electric effect to transmit a small crystal lattice distortion to the tip of a pipette, driving it forward in a precise and controlled manner. Piezo-actuated micromanipulation has multiple applications in the study and engineering of gametes and embryos. It enabled the first intracytoplasmic sperm injection (ICSI) to produce mice 1 , the first nuclear transfer cloning of mice 2 and pigs 3 , the first productive frozen 4 and freeze-dried sperm injections 5 , and the first production of nuclear transfer embryonic stem (ES) cells 6 . Piezo was utilized to generate the first transgenic offspring by injecting unfertilized oocytes in metaphase II (mII) transgenesis 7 and has been extended to the delivery of artificial chromosome transgenes 8,9 . Piezo has been employed for RNA interference (RNAi) in mII oocytes 10 ; it enhances blastocyst injection with ES cells 11 and the manipulation of gamete precursors 12 and facilitates the renaissance of stem cell biology 13 .Piezo-actuated micromanipulation was developed by Atsushi Mimatsu and colleagues, and its application as a biological research tool was demonstrated in the mouse by Dr. Yasuyuki Kimura, who used it to generate the first mouse offspring by ICSI 1 . Piezo was necessary because in the mouse, oocyte plasma membranes are exquisitely sensitive and survival rates following ICSI with conventional (i.e., manual, non-piezo) microinjection rarely exceed 50% (ref. 14). Contrastingly, piezo ICSI can achieve mouse oocyte survival rates of 100%, with 90% development to morula/blastocyst stages in vitro. The efficacy of piezo is highly desirable, not only in ICSI but also where development following microinjection is less efficient, such as in nuclear transfer. Most mouse nuclear transfer

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Pig Cloning by Microinjection of Fetal Fibroblast Nuclei

Cited by 790 publications

References 21 publications

Development of Human‐Like Advanced Coronary Plaques in Low‐Density Lipoprotein Receptor Knockout Pigs and Justification for Statin Treatment Before Formation of Atherosclerotic Plaques

Development of Human‐Like Advanced Coronary Plaques in Low‐Density Lipoprotein Receptor Knockout Pigs and Justification for Statin Treatment Before Formation of Atherosclerotic Plaques

Pig transgenesis by Sleeping Beauty DNA transposition

Piezo-actuated mouse intracytoplasmic sperm injection (ICSI)

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