Direct Comparison between ICSI-Mediated DNA Transfer and Pronuclear DNA Microinjection for Producing Transgenic Rats

Hirabayashi, Masumi; Kato, Megumi; Amemiya, Kazue; Hochi, Shinichi

doi:10.1538/expanim.57.145

Cited by 8 publications

(7 citation statements)

References 15 publications

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“…ICSI has been used to circumvent problems of penetration/fertilisation by the sperm. ICSI can also be used to co-inject sperm and DNA into the oocyte, as a way of achieving transgenesis, (Perry et al 1999) with similar efficiency to pronuclear microinjection; this method has proved successful not only in mice but also in rats (Hirabayashi et al 2008). This technique has been also reported to produce YAC transgenics (Moreira et al 2004) with an efficiency 10 times greater than that usually obtained by standard microinjection.…”

Section: Transgenesis By Icsimentioning

confidence: 99%

The COST Manual of Laboratory Animal Care and Use

Howard¹,

Nevalainen²,

Perretta³

2016

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The Open Access version of this book, available at www.taylorfrancis.com, has been made available under a Creative Commons Attribution-Non Commercial-No Derivatives 4.0 license.Neither the COST Office nor any person acting on its behalf is responsible for the use which might be made of the information contained in this publication. The COST Office is not responsible for the external websites referred to in this publication.

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Section: Transgenesis By Icsimentioning

confidence: 99%

The COST Manual of Laboratory Animal Care and Use

Howard¹,

Nevalainen²,

Perretta³

2016

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“…Embryo culture technique is not well adapted for use in rats Dann 2007 Injected DNA often results in random insertion of concatamers Hirabayashi et al 2008Popova et al 2008 (2) Microinjection using an artificial chromosome vector Cloning capacity of the chromosome vector is more than 1 Mb With this technique transgenic animals carry single or few copies of the transgene Sperm-mediated DNA transfer is based on the ability of sperm cells to bind and internalize exogenous DNA (thus, they can act as vector for exogenous DNA) and transfer it into the egg during fertilization under in vitro or in vivo conditions (Lavitrano et al 2006, Smith andSpadafora. 2005).…”

Section: Filipiak and Saunders 2006mentioning

confidence: 99%

Beyond the Rat Models of Human Neurodegenerative Disorders

2009

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The rat is a model of choice in biomedical research for over a century. Currently, the rat presents the best "functionally" characterized mammalian model system. Despite this fact, the transgenic rats have lagged behind the transgenic mice as an experimental model of human neurodegenerative disorders. The number of transgenic rat models recapitulating key pathological hallmarks of Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, or human tauopathies is still limited. The reason is that the transgenic rats remain more difficult to produce than transgenic mice. The gene targeting technology is not yet established in rats due to the lack of truly totipotent embryonic stem cells and cloning technology. This extremely powerful technique has given the mouse a clear advantage over the rat in generation of new transgenic models. Despite these limitations, transgenic rats have greatly expanded the range of potential experimental approaches. The large size of rats permits intrathecal administration of drugs, stem cell transplantation, serial sampling of the cerebrospinal fluid, microsurgical techniques, in vivo nerve recordings, and neuroimaging procedures. Moreover, the rat is routinely employed to demonstrate therapeutic efficacy and to assess toxicity of novel therapeutic compounds in drug development. Here we suggest that the rat constitutes a slightly underestimated but perspective animal model well-suited for understanding the mechanisms and pathways underlying the human neurodegenerative disorders.

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“…The production efficiency of transgenic rats by the PNMI-tg and ICSI-tg methods have been directly compared using six DNA constructs [21]. No significant difference was found in the production efficiency of PCR-positive transgenic rats between the ICSI-tg and PNMI-tg methods by a non-parametric paired t-test (p=0.22; DNA code-A 1.1 vs. 0.9%, code-B 1.3 vs. 0.2%, code-C 3.1 vs. 2.4%, code-D 1.7 vs. 0.5%, code-E 0.2 vs. 2.2%, code-F 0.2 vs. 0%, respectively).…”

Section: Successful Application To Rat Transgenesismentioning

confidence: 99%

“…2). Due to the larger surface area of the sperm heads of rats compared with mice, exposure to a higher concentration of exogenous DNA may lead to excess DNA association to the sperm heads and introduction into oocytes by ICSI, which may have a toxic effect on development of oocytes derived from the ICSI-tg method.The production efficiency of transgenic rats by the PNMI-tg and ICSI-tg methods have been directly compared using six DNA constructs [21]. No significant difference was found in the production efficiency of PCR-positive transgenic rats between the ICSI-tg and PNMI-tg methods by a non-parametric paired t-test (p=0.22; DNA code-A 1.1 vs. 0.9%, code-B 1.3 vs. 0.2%, code-C 3.1 vs. 2.4%, code-D 1.7 vs. 0.5%, code-E 0.2 vs. 2.2%, code-F 0.2 vs. 0%, respectively).…”

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confidence: 99%

Technical Development for Production of Gene-Modified Laboratory Rats

Hirabayashi

2008

Journal of Reproduction and Development

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Abstract. Transgenic rats have been used as model animals for human diseases and organ transplantation and as animal bioreactors for protein production. In general, transgenic rats are produced by pronuclear microinjection of exogenous DNA. Improvement of post-injection survival has been achieved by micro-vibration of the injection pipette. The promoter region, structural gene, chain length and strand ends of the exogenous DNA are not involved in the production efficiency of transgenic rats. Exogenous DNA prepared at 5 μg/ml seemed to be better integrated than lower and higher concentrations. Intracytoplasmic sperm injection (ICSI) has been successfully achieved in rats using a piezo-driven injection pipette. The ICSI technique has not only been applied to rescue infertile male strains but also to produce transgenic rats. The optimal DNA concentration for the ICSI-tg method (0.1 to 0.5 μg/ml) is lower than that for the conventional pronuclear microinjection. Production efficiency was improved when the membrane structure of the sperm head was partially disrupted by detergent or ultrasonic treatment before exposure to the exogenous DNA solution. For successful production of transgenic rats with a modified endogenous gene, establishment of embryonic stem cell lines or alternatively male germline stem cell lines and technical development of somatic cell nuclear transfer are still necessary for this species. Key words: DNA microinjection, Intracytoplasmic sperm injection (ICSI)-mediated transgenesis, Transgenic rat (J. Reprod. Dev. 54: [95][96][97][98][99] 2008) ransgenic rats have been used as model animals for human diseases (e.g., Alzheimer's disease, autoimmunity and highdensity lipoprotein metabolism) [1][2][3] and organ transplantation [4] and as animal bioreactors for protein production [5,6]. The advantage of using rats in transgenic studies would be the ease of continuous or repeated collection of secreted samples (e.g., blood or urine) and various surgical treatments due to the larger size of rats compared with mice, while the litter size, gestation period, maturation rate, estrus cycle length and lifespan of rats are almost comparable with those of mice. Thus, the rat has the advantage of being a somewhat-characterized and intermediate-sized rodent without the economic disadvantage of larger animals and without the technical disadvantage of smaller rodents. Successful production of transgenic rats was first reported at three independent laboratories [2,7,8] by pronuclear microinjection of exogenous DNA, the most convenient and reproducible technique of producing transgenic animals. In addition, exogenous DNA has been introduced into the mouse and pig genomes using spermatozoa as the vector under a conventional IVF technique [9,10]. While the reproducibility of the sperm-mediated DNA transfer technology is still questionable [11,12], it has been reported that transgenic mice can be produced when sperm heads and an exogenous DNA solution are mixed and co-injected into oocytes using the ICSI technique [13]...

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Direct Comparison between ICSI-Mediated DNA Transfer and Pronuclear DNA Microinjection for Producing Transgenic Rats

Cited by 8 publications

References 15 publications

The COST Manual of Laboratory Animal Care and Use

The COST Manual of Laboratory Animal Care and Use

Beyond the Rat Models of Human Neurodegenerative Disorders

Technical Development for Production of Gene-Modified Laboratory Rats

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