Pronuclear Microinjection

Wall, Robert J.

doi:10.1089/15362300152725936

Cited by 83 publications

(77 citation statements)

References 75 publications

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“…Further studies will entail generation of a diabetic model pig using the PD2 construct assessed in this study. Despite the fact that microinjection of exogenous DNA into male pronuclei has proven to be highly successful in the generation of transgenic animals, in mice particularly as shown in this study, this technique has proven to be far less efficient in farm animals [42], including the pig. Recently, gene transfer using intra-cytoplasmic sperm injection (ICSI-mediated gene transfer) and in vitro-matured oocytes has been reported as an efficient alternative procedure for producing transgenic pigs [43].…”

Section: Discussionmentioning

confidence: 79%

The Production of a Diabetic Mouse Using Constructs Encoding Porcine Insulin Promoter-Driven Mutant Human Hepatocyte Nuclear Factor-1.ALPHA.

Watanabe¹,

Umeyama²,

Kawano

et al. 2007

Journal of Reproduction and Development

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Abstract. A diabetic mouse model was produced using a mutant human hepatocyte nuclear factor-1α gene (HNF1αP291fsinsC) regulated by the porcine insulin promoter. The functionality of two different constructs containing HNF1αP291fsinsC, termed PD1 and PD2 (cytomegalovirus enhancer minus and plus), were examined in transgenic mice. The blood glucose levels and body weights of the PD1 transgenic mice did not differ from their non-transgenic littermates over the period from 3 to 8 weeks of age. Conversely, the PD2 transgenic mice exhibited hyperglycemia and decreased body weight. Western blot analysis demonstrated that mutant HNF-1α protein (HNF1αP291), derived from the PD2 transgene, was expressed in the PD2 mice. Morphometric studies of the pancreas of a PD2 mouse revealed that the number of pancreatic islets present was less than that in the nontransgenic mice, indicating disturbed islet neogenesis. These results suggest that impaired insulin secretion in disrupted islets causes hyperglycemia. In addition, the phenotype of PD2 transgenic mice similar to that of the HNF-1α gene-deficient mouse, which displays growth retardation and impaired viability. These results indicate that HNF1αP291 expression driven by the porcine insulin promoter, together with the cytomegalovirus enhancer, induces a diabetic phenotype in transgenic mice.

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Section: Discussionmentioning

confidence: 79%

The Production of a Diabetic Mouse Using Constructs Encoding Porcine Insulin Promoter-Driven Mutant Human Hepatocyte Nuclear Factor-1.ALPHA.

Watanabe¹,

Umeyama²,

Kawano

et al. 2007

Journal of Reproduction and Development

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“…Transgenic animals have, for the most part, been produced using the microinjection of exogenous DNA into the male pronuclei of zygotes (Wall 2001). This technique, although highly successful in mice, is not as efficient in farm animals, a fact that limits its general usefulness.…”

Section: Production Of Transgenic Pigsmentioning

confidence: 99%

“…Over the past 25 years, a variety of methods that allow the generation of transgenic animals have been developed (Wall 2001(Wall , 2002 and the first transgenic livestock were born 20 years ago (Hammer et al 1985). Today, the most widely used methods for the production of transgenic farm animals are: (1) direct microinjection of foreign DNA into the pronuclei of fertilised eggs; (2) nuclear transfer using genetically modified embryonic or somatic donor cells; and (3) viral-based constructs as vectors for the introduction of exogenous DNA into embryos (Kues and Niemann 2004).…”

Section: Introductionmentioning

confidence: 99%

Sperm-mediated gene transfer

Lavitrano

Busnelli

Cerrito

et al. 2006

Reprod. Fertil. Dev.

115

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Abstract. Since 1989, a new method for the production of transgenic animals has been available, namely spermmediated gene transfer (SMGT), based on the intrinsic ability of sperm cells to bind and internalise exogenous DNA molecules and to transfer them into the oocyte at fertilisation. We first described the SMGT procedure in a small animal model, with high efficiency reported in the mouse. In addition, we successfully adapted and optimised the technique for use in large animals; it was, in fact, highly efficient in the generation of human decay accelerating factor transgenic pig lines, as well as multigene transgenic pigs in which three different reporter genes, namely enhanced green fluorescent protein, enhanced blue fluorescent protein and red fluorescent protein, were introduced. The major benefits of the SMGT technique were found to be its high efficiency, low cost and ease of use compared with other methods. Furthermore, SMGT does not require embryo handling or expensive equipment. Sperm-mediated gene transfer could also be used to generate multigene transgenic pigs that would be of benefit as large animal models for medical research, for agricultural and pharmaceutical applications and, in particular, for xenotransplantation, which requires extensive genetic manipulation of donor pigs to make them suitable for grafting to humans.

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“…The transgenic methods in use today were developed in the past 25 years and these traditional methods of genetic engineering and transgenesis insert genes at random locations within the large genome of higher organisms, resulting in loss of efficiency, unpredictable results, and unintended genetic consequences (Perry et al 1999;Wall 2001;Lois et al 2002;Wall 2002). The pronuclear microinjection technique was the first to be conceived and was developed specifically to produce germline transgenic mice.…”

Section: Introductionmentioning

confidence: 99%

“…The pronuclear microinjection technique was the first to be conceived and was developed specifically to produce germline transgenic mice. It has generated transgenic animals in a wide variety of mammalian species, usually with multiple concatemerised vector copies (Muller 1999;Wall 2001).…”

Section: Introductionmentioning

confidence: 99%

Active integration: new strategies for transgenesis

Shinohara

Kaminski²,

Segal

et al. 2007

Transgenic Res

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This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

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Pronuclear Microinjection

Cited by 83 publications

References 75 publications

The Production of a Diabetic Mouse Using Constructs Encoding Porcine Insulin Promoter-Driven Mutant Human Hepatocyte Nuclear Factor-1.ALPHA.

The Production of a Diabetic Mouse Using Constructs Encoding Porcine Insulin Promoter-Driven Mutant Human Hepatocyte Nuclear Factor-1.ALPHA.

Sperm-mediated gene transfer

Active integration: new strategies for transgenesis

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