Microinjection: Methods and Applications

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This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination.

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To increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.

For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation F1 of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras.

When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation. Transgenic animals are just one in a series of developments in the area of biotechnology. Biotechnology has transformed the way in which we understand processes such as engineering and manufacturing.

These terms now include the use of living organisms or their parts to make or modify products, to change the characteristics of plants or animals, or to develop micro-organisms for specific uses. The novel uses of biological techniques such as recombinant DNA techniques, cell fusion techniques, mono and polyclonal antibody technology and biological processes for commercial production have altered traditional distinctions and methods US Congress, Office of Technology Assessment, Genetic manipulations at the level of DNA have also changed long held views as to what is considered to be animal, plant and human.

In turn, these changes have made it more difficult to evaluate the ways in which animals are used and have obscured distinctions between pure and applied research. Consideration of the acceptability of creating specific transgenic animal strains or genetic manipulation involving interchanging DNA between species and kingdoms could be a simple animal care issue or a societal decision. The following is an attempt to show what the ability to create transgenic animals or engage in other forms of DNA manipulation means in terms of traditional ACC functions, not forgetting that this impacts on wider considerations of human responsibility for the welfare of other life forms.

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The creation of transgenic animals is resulting in a shift from the use of higher order species to lower order species, and is also affecting the numbers of animals used. An example of the replacement of higher species by lower species is the possibility to develop disease models in mice rather than using dogs or non-human primates.

In the long term, a reduction in the number of animals used, for example to study human diseases, is possible due to a greater specificity of the transgenic models developed. On the other hand, the success of the method has led to using its potential for investigating a wider range of diseases and conditions. The actual use of some species may be increased, in addition to the numbers of animals which are sacrificed as donors during the creation process. The potential of the technology has also made it possible to consider employing cattle, swine, sheep and goats as processing units to manufacture proteins or as organ donors.

The complex interactive processes of living mammals are not reproducible in vitro. However, transgenic animals provide a means of evaluating genetic modifications in terms of anatomical and physiological changes in a complex system. Transgenic models are more precise in comparison to traditional animal models, for example the oncomouse with its increased susceptibility to tumor development enables results for carcinogenicity studies to be obtained within a shorter time-frame, thus reducing the course of tumor development in experimentally affected animals. However, models are not strict equivalents, so as with any other system care must be taken in drawing conclusions from the data.

A representative, but non-inclusive, list of purposes for which transgenic animals have been used indicates the wide ranging application of this biotechnology:. Important general considerations include the extent to which experience acquired in the laboratory with regard to husbandry should influence industry standards for keeping animals created specifically as living machines for the production of proteins, antibodies, etc. What words are appropriate to describe and evaluate the condition of animals now used as production units? The successful cloning of Dolly underlines the fact that innovative developments in animal science are part of the mainstream of biotechnology.

In addition, the use of xenografts, at least at the public health level makes animal and human welfare inseparable. For animal welfare concerns regarding the use of transgenic animals, see Transgenic Animals, Animal Welfare and Ethics optional. For a practical application of transgenics, see Transgenic Goats optional.

Medical applications of transgenics optional. The effect of cells transferred into mouse blastocyst on subsequent development. Donnelly, S. In Chapter 6, a practical method for microinjection, and subsequent insemination, of the unfertilized zebrafish egg is reported. This will open up more opportunities for exploiting an already useful model system. The chapter is very detailed which should allow these methods to be adapted to many different situations.

The chapter also features a very extensive and useful notes section, detailing specifics of each of the methods.

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Methods for developing recombinant cells lines by microinjection into the nucleus are presented in Chapter 8. This very interesting article demonstrates that introduction of plasmid DNA for the GFP, in several cell types, leads to stable transduction as assayed by flow cytometry of GFP fluorescence up to 1 month after microinjection! The chapter details methods that could be adapted to virtually any cell type and for any DNA. Conditions for optimizing the production of the transformed cells lines are tested in this chapter and detailed notes are given to help any investigator interested in attempting this method.

Different experimental methods, including transposons, I-SceI meganuclease, and direct injection of linearized DNA, have been used to produce transgenic Xenopus for the study of a variety of problems 24— However, these methods rely upon random insertion into the Xenopus genome or produce multiple copies. In Chapter 9, a technique is described for the targeted insertion of a single copy of the gene of interest into Xenopus laevis using phiC31 integrase.

By incorporating insulator sequences into the plasmid design, they improve expression from the reporter gene making this method extremely useful for anyone wishing to express a transgene at approximately endogenous levels. Transgenics are also the topic of Chapter A method for producing transgenic Caenorhabditis elegans by microinjecting DNA directly into the hermaphrodite gonad is provided. For those not familiar with C.

It recounts the basic reproductive biology of the worm and includes a discussion of applications for microinjection. Techniques for cultivating the worms and special hints for maximizing success of microinjection are explained in detail.

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One of the great advantages of using microinjection to introduce molecules into living cells is the ability to perform quantitative experiments. Chapter 11 gives detailed methods for the quantitative microinjection of picoliter quantities into mouse oocytes and eggs in dishes on an inverted microscope. The technique is described in wonderful detail and covers all aspects from making the glass bottom dishes that hold the oocytes to the injection process itself. There are several items of note in the chapter.

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I particularly enjoyed learning how to use an old record player to construct a beveler capable of producing a nice 1—2 mm tip. But then, what will I do with my old Pink Floyd records? Two methods are given in Chapter 11 to achieve this: 1 using mercury to backfill the micropipette and relying upon the mercury to transduce and control pressure and 2 constructing a microneedle with a constriction near the tip which reduces fluid movement and provides fine control of the injection.

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Both types of microinjection needles can be used in the same overall system. A similar microinjection system is described in Chapter 12, with modifications that allow for injection of the mouse oocyte within the complete follicle. Because the mouse oocyte exists within the follicle when in the ovary, it is intimately associated with the surrounding follicle cells until after ovulation.

This relatively new method allows for the microinjection to occur while the oocyte is cultured within the intact follicle under more physiologically relevant conditions.

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By microinjecting the follicle-enclosed oocytes, this group has been able to discover the mechanism s that maintain the immature oocyte arrested in meiosis and also to begin exploring the signaling mechanisms that are responsible for the reinitiation of meiosis 27— The technique is quite revolutionary and the concept of maintaining the proper physiological environment, as much as possible, is one that is applicable to all situations.

In this chapter, you will also learn what type of music is most enjoyed by mouse oocytes. In some cases, microinjection would be very useful but not considered because of the perceived difficulty of injecting sufficient numbers of cells for further analysis. Chapter 13 describes a pressure-based method of injecting zygotes of the sea urchin Paracentrotus lividus. Using this method, up to several hundred embryos can be injected in a single session. In addition to the microinjection method, this chapter also describes experimental methods for perturbing gene function by either 1 producing and microinjecting synthetic mRNA produced in vitro, or 2 preparing linear DNA amplified by PCR for direct microinjection into the embryo.

The advantages and disadvantages of these two different methods are discussed.

Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications Microinjection: Methods and Applications
Microinjection: Methods and Applications

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