Germ Line Transformation of Mammals by Pronuclear Microinjection

Rülicke, Thomas; Hübscher, Ulrich

doi:10.1111/j.1469-445x.2000.02092.x

Cited by 27 publications

(15 citation statements)

References 51 publications

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“…When it reaches the 2-cell stage, the embryo is reimplanted into the uterus of a pseudopregnant female. Depending on the skill of the research worker, the success rate of this technique ranges from approximately 1% to 8% of the total number of microinjected eggs [12][13][14][15][16][17]. The second approach involves the in vitro transfection of embryonic stem (ES) cells [18].…”

Section: Introductionmentioning

confidence: 99%

The Making of “Transgenic Spermatozoa”1

Célébi

Guillaudeux

Auvray

et al. 2003

Biology of Reproduction

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The processes of making transgenic animals by microinjecting DNA into the pronucleus of a fertilized oocyte or after the transfection of embryonic stem cells are now well established. However, attempts have also been made, with varying degrees of success, to use spermatozoa as a vector for transgenesis in mammals and other vertebrates during the last decade. A number of different approaches for making transgenic spermatozoa have been developed. These include directly incubating mature, isolated spermatozoa with DNA or pretreating mature, isolated spermatozoa before assisted fertilization. Microinjection procedures have also been established to transfect male germ cells directly in vivo within the seminiferous tubules or to reimplant previously isolated male germ cells submitted to in vitro transfection into a recipient testis. The latter two techniques present the advantage of being able to create transgenic progeny simply by mating with wild-type females, which avoids the possibility of interference or damage as a result of assisted fertilization or the manipulation of embryos. The different aspects of sperm-mediated transgenesis are presented.

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Section: Introductionmentioning

confidence: 99%

The Making of “Transgenic Spermatozoa”1

Célébi

Guillaudeux

Auvray

et al. 2003

Biology of Reproduction

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“…The number of copies of plasmid DNA that can be integrated varies from one to several hundred copies, which can be found as concatemers with a predominant head-to-tail orientation (3,4,77). Inhibitory effects from nearby genomic areas can alter the expression of the transgene (1), which is why larger genomic fragments are often used that include insulator sequences to maximize the production of transgene-expressing transgenic animals (113). The unrestricted size limitation is the single most important advantage to creating transgenic animals through direct incorporation of plasmid DNA.…”

mentioning

confidence: 99%

Lentiviral vectors: are they the future of animal transgenesis?

Park

2007

Physiological Genomics

148

103

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Lentiviral vectors have become a promising new tool for the establishment of transgenic animals and the manipulation of the mammalian genome. While conventional microinjection-based methods for transgenesis have been successful in generating small and large transgenic animals, their relatively low transgenic efficiency has opened the door for alternative approaches, including lentiviral vectors. Lentiviral vectors are an appealing tool for transgenesis in part because of their ability to incorporate into genomic DNA with high efficiency, especially in cells that are not actively dividing. Lentiviral vector-mediated transgene expression can also be maintained for long periods of time. Recent studies have documented high efficiencies for lentiviral transgenesis, even in animal species and strains, such as NOD/scid and C57Bl/6 mouse, that are very difficult to manipulate using the standard transgenic techniques. These advantages of the lentiviral vector system have broadened its use as a gene therapy vector to additional applications that include transgenesis and knockdown functional genetics. This review will address the components of the lentiviral vector system and recent successes in lentiviral transgenesis using both male- and female-derived pluripotent cells. The advantages and disadvantages of lentiviral transgenesis vs. other approaches to produce transgenic animals will be compared with regard to efficiency, the ability to promote persistent transgene expression, and the time necessary to generate a sufficient number of animals for phenotyping.

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“…The classic approaches include nerve section, vascular occlusion or removal of an organ. However, in this post-genomic era powerful new techniques have become available including gene knockout, conditional knockouts, transgenesis, somatic gene transfer and antisense technology, which allow an examination of integrative responses to single gene alterations (Bader et al 2000;Culman, 2000;Kasparov & Paton, 2000;Ledermann, 2000;Rulicke & Hubscher, 2000).…”

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confidence: 99%

Combined Confocal Microscopy and Stereology: a Highly Efficient and Unbiased Approach to Quantitative Structural Measurement in Tissues

Howell

Hopkins

McLoughlin

2002

Experimental Physiology

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Understanding the relationship of the structure of organs to their function is a key component of integrative physiological research. The structure of the organs of the body is not constant but changes, both during growth and development and under conditions of sustained stress (e.g. high altitude exposure and disease). Recently, powerful new techniques have become available in molecular biology, which promise to provide novel insights into the mechanisms and consequences of these altered structure‐function relationships. Conventionally structure‐function relationships are studied by microscopic examination of tissue sections. However, drawing conclusions about the three‐dimensional structure of an organ based on this two‐dimensional information frequently leads to serious errors. The techniques of stereology allow precise and accurate quantification of structural features within three‐dimensional organs that relate in a meaningful way to integrated function. For example, knowledge of changes in the total surface area of the capillary endothelium in an organ can be related directly to changes in fluid filtration and permeability, or knowledge of total vessel length and mean radius allows deductions about vascular resistance. Confocal microscopy adds enormously to the power of stereological approaches. It reduces the difficulties and labour involved in obtaining suitable images. Moreover, when used in conjunction with new analytical software, it allows convenient application of stereology to small samples and those in which it is essential to maintain a specific orientation for interpretation. The information obtained will allow us to examine in a quantitative manner the altered structure‐function relationships produced by manipulation of single genes and regulatory pathways in whole organisms.

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Germ Line Transformation of Mammals by Pronuclear Microinjection

Abstract: 591Figure 2Development of pronuclei and microinjection of DNA into the mouse zygote. A and C, development of pronuclei (arrows), about 20 and 26 h post hCG-injection for superovulation; B, DNA-microinjection into the male pronucleus; D, pronuclei disappear before the first cell division.

Cited by 27 publications

References 51 publications

The Making of “Transgenic Spermatozoa”1

The Making of “Transgenic Spermatozoa”1

Lentiviral vectors: are they the future of animal transgenesis?

Combined Confocal Microscopy and Stereology: a Highly Efficient and Unbiased Approach to Quantitative Structural Measurement in Tissues

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