Microinjection: Methods and Applications (Methods in Molecular Biology Vol. 518)

Edited by David J. Carroll

Humana Press (2009) 224 pages, ISBN 978-1-58829-884-3, $99.50 (hardback) Microinjection: Methods and Applications (Methods in Molecular Biology Vol. 518) Edited by David J. Carroll Humana Press (2009) 224 pages, ISBN 978-1-58829-884-3,$99.50 (hardback)

This is just the type of book I would have liked to have had read before I began my own injection studies in the early 1980s. In Microinjection: Methods and Applications, David Carroll, the Editor, has assembled a knowledgeable group of practitioners, who describe clearly how to inject molecules, nuclei and cells (sperm) into a range of target cells. The difference between injection then and now, however, is not really how to inject, but what to inject and how to analyze the effects of such manipulations. Again, the chapter authors do a good job of describing various analytical methods, often in great detail.

Cellular injection, which ranges from the injection of somatic cells growing in vitro, to injecting into much larger cells, such as fertilized eggs, has a long history. Its roots are in the work of early cell biologists, perhaps best epitomized by Robert Chambers (1881-1957), who used a range of microknives and microneedles to probe the physiochemical nature of the living state. The design and fabrication of improved micromanipulators made possible a wide range of subtle perturbations of cells and intracellular structures (see Chambers, 1918; Chambers, 1922). The best-appreciated ‘neoclassical’ examples of such techniques include the pioneering studies of Briggs and King (Briggs and King, 1952) and Sir John Gurdon (Gurdon, 1960; Gurdon and Byrne, 2003), who used nuclear transplantation to explore the nature of the epigenetic changes associated with embryonic development, and those of R. B. Nicklas (Nicklas and Staehly, 1967; Nicklas, 1967) and others, who used micromanipulation to characterize the mechanical and adaptive properties of the eukaryotic spindle. Injection and cellular dissection methods were also key to defining the properties of nuclear-cytoplasmic targeting systems (Goldstein and Prescott, 1967; Paine and Feldherr, 1972).

The ‘modern’ age of cellular manipulation began with the introduction of more versatile and specific tracers and perturbants, including fluorescently labelled proteins (Taylor and Wang, 1978) and function-blocking antibodies. Using the injection of an anti-myosin antisera, Mabuchi and Okuno resolved the role of actin-myosin in cytokinesis (as distinct from mitosis) (Mabuchi and Okuno, 1977). Shortly thereafter the intracellular injection of monoclonal antibodies was found to be the first specific method to disrupt intermediate filament organization (Lin and Feramisco, 1981; Klymkowsky, 1981; Klymkowsky, 1982). Since then, many new experimental reagents have become available, including anti-sense and microRNA-based approaches, together with the expression of wild-type, epitope-tagged, and engineered polypeptides.

In my own case, mastering injection involved assembling an injection system (including a customized stand to hold a Leitz micromanipulator, and a large bore glass syringe and heavy rubber bands as a ‘controlled’ pressure source). These days there are many commercially available injection systems that significantly lower the activation energy involved in beginning and successfully completing such studies. Once convinced that mammalian cells could survive injection (by me), experiments became routine (aside from the frustration of clogged needles). While each investigator has to have their own ‘conversion experience’, they will be greatly helped by the clarity of the presentations provided in Microinjection: Methods and Applications.

The book is organized into 13 chapters that cover the injection of oocytes, eggs, embryos and germ lines in C. elegans, sea urchin, starfish, frog, zebrafish, mouse and human. Each chapter deals with a particular organism and a particular experimental manipulation. For example, the four chapters on the clawed frog Xenopus laevis are focused on mRNA and antisense (morpholino) oligonucleotide injection in embryos, mRNA injection/protein expression in oocytes, and the generation of transgenic organisms. Each chapter contains a number of helpful illustrations, as well as a ‘notes’ section that addresses specific experimental issues. The book's audience, primarily novices who seek guidance (and some reassurance) on how to make this experimental approach work for them, should find it quite helpful. One useful addition would be the inclusion of more videos (such as those referenced in Douglas Kline's chapter). I know from my own experience with students that showing them (by using a video system) exactly what injection looks like greatly reduces the time it takes for them to become proficient injectors.

This is just the type of book I would have liked to have had read before I began my own injection studies in the early 1980s

While the chapters are uniformly thorough, I did find myself taking exception to, or wanting more discussion about, some points — my comments are restricted to the six chapters on mammalian cells and Xenopus, the systems with which I have direct experience. In contrast to what is stated in the chapters on mammalian cell injection, only a few of the commonly studied cell types require glass cover slips to be treated to ensure the cells adhere to them, and there are simpler methods to hold cells during injection than Rose chambers. More importantly, there is no justification for the suggestion that miRNA-type reagents are, per se, more or less specific than injected antibodies. While an antibody can exhibit ‘cross-recognition of other proteins within the cell’ (p. 78), RNA reagents can also display off-target effects (Qiu et al., 2005). With respect to intranuclear injection, I have often wondered, while watching the flow of injected solutions, whether it might lead to breaks in genomic DNA, and as such had hoped that in the chapter written by Sebastien Chenuet et al. this possibility would have been discussed.

Similarly, my concerns with respect to the four chapters on Xenopus are largely minor and technical. For example, in Chapter 1 (written by Jill Sible and Brian Wroble), I was surprised by the absence of a discussion of hormone-regulated proteins (delivered by RNA injection). This method, introduced by Hollenberg et al. (Hollenberg et al., 1993) and popularized by Kolm and Sive (Kolm and Sive, 1995), is simple, works with many different types of proteins, including non-transcription factors (Zhang et al., 2006), and together with targeted blastomere injection allows the investigator to control where and when a protein becomes active. Combined with the use of protein synthesis inhibitors, it provides initial (albeit not definitive) evidence about whether a regulatory interaction is direct or indirect. On a related note, while these authors recommend a commercial source for the pSP64T plasmid, they fail to note that other plasmids are readily obtained from members of the Xenopus community through the XenBase web resource. Given the high level of experimental detail in this and other chapters, one might have expected discussions of: (1) the typical (that is, reasonable) amounts of antibodies, RNAs, DNAs and morpholinos to be injected; (2) the use of GFP-chimeras to monitor RNA-directed expression and morpholino activity; and, importantly, (3) the fact that injection into a single blastomere does not lead to a uniform and global distribution of the injected molecule or its product. Particularly in the case of polypeptide encoding RNAs, post-injection diffusion can be quite limited, presumably because of interactions with ribosomes — typically uniform expression requires injection of both blastomeres at the two-cell stage, or of all four blastomeres at the four-cell stage, something that can be readily demonstrated through whole-mount immunocytochemistry.

In Chapter 3 (by Jeffrey Lau and Anthony Muslin), the authors describe the use of immunoblot analysis to characterize the effects of translation blocking morpholinos. They probably should have noted: (1) that such morpholinos have been reported to stabilize target RNAs (Gene-Tools web site and our own experimental observations), which can make in situ hybridization and RT-PCR analysis methods problematic; and (2) that immunoblot relies on the availability of a good antibody against the target protein. Alternatives are available; for example, one can use in vitro translation systems or RNAs that contain the target sequence and encode an epitope-tagged version of the protein (see Zhang et al., 2006). Conversely, if RNA splice-blocking morpholinos are used, RT-PCR analysis is straightforward. Finally, the method for generating transgenic X. laevis embryos described by Bryan Allen and Daniel Weeks (Chapter 9) is clearly exciting, but it is worth noting that since it was first described in 2005 (Allen and Weeks, 2005) no other lab has (apparently) published using the technique. In this light, a general review of other transgenic methods (Ogino et al., 2006; Waldner et al., 2006; L'Hostis-Guidet et al., 2009), as well as a discussion of the application of these methods to X. tropicalis, a diploid relative with a shorter generation time and a sequenced genome, would seem to be both appropriate and useful.

All of which is to say that, while this book provides a useful guide to experimental design, the savvy investigator should take the time to explore the various ancillary technical issues associated with their specific project.

Allen
B. G.
,
Weeks
D. L.
(
2005
).
Transgenic Xenopus laevis embryos can be generated using phiC31 integrase
.
Nat. Methods
2
,
975
-
979
.
Briggs
R.
,
King
T. J.
(
1952
).
Transplantation of living nuclei from blastula cells into enucleated frogs' eggs
.
38
,
455
-
463
.
Chambers
R.
(
1918
).
The microvivisection method
.
Biol. Bull.
34
,
121
-
136
.
Chambers
R.
(
1922
).
New apparatus and methods for the dissection and injection of living cells
.
Anat. Rec.
24
,
1
-
19
.
Goldstein
L.
,
Prescott
D. M.
(
1967
).
Proteins in nucleocytoplasmic interactions. I. The fundamental characteristics of the rapidly migrating proteins and the slow turnover proteins of the Amoeba proteus nucleus
.
J. Cell Biol.
33
,
637
-
644
.
Gurdon
J. B.
(
1960
).
Factors responsible for the abnormal development of embryos obtained by nuclear transplantation in Xenopus laevis
.
J. Embryol. Exp. Morphol.
8
,
327
-
340
.
Gurdon
J. B.
,
Byrne
J. A.
(
2003
).
The first half-century of nuclear transplantation
.
100
,
8048
-
8052
.
Hollenberg
S. M.
,
Cheng
P. F.
,
Weintraub
H.
(
1993
).
Use of a conditional MyoD transcription factor in studies of MyoD trans-activation and muscle determination
.
90
,
8028
-
8032
.
Klymkowsky
M. W.
(
1981
).
Intermediate filaments in 3T3 cells collapse after intracellular injection of a monoclonal anti-intermediate filament antibody
.
Nature
291
,
249
-
251
.
Klymkowsky
M. W.
(
1982
).
Vimentin and keratin intermediate filament systems in cultured PtK2 epithelial cells are interrelated
.
EMBO J.
1
,
161
-
165
.
Kolm
P. J.
,
Sive
H. L.
(
1995
).
Efficient hormone-inducible protein function in Xenopus laevis
.
Dev. Biol.
171
,
267
-
272
.
L'Hostis-Guidet
A.
,
Recher
G.
,
Guillet
B.
,
A.
,
Coumailleau
P.
,
Tiaho
F.
,
Boujard
D.
,
T.
(
2009
).
Generation of stable Xenopus laevis transgenic lines expressing a transgene controlled by weak promoters
.
Transgenic Res.
18
,
815
-
827
.
Lin
J. J.
,
Feramisco
J. R.
(
1981
).
Disruption of the in vivo distribution of the intermediate filaments in fibroblasts through the microinjection of a specific monoclonal antibody
.
Cell
24
,
185
-
193
.
Mabuchi
I.
,
Okuno
M.
(
1977
).
The effect of myosin antibody on the division of starfish blastomeres
.
J. Cell Biol.
74
,
251
-
263
.
Nicklas
R. B.
(
1967
).
Chromosome micromanipulation. II. Induced reorientation and the experimental control of segregation in meiosis
.
Chromosoma
21
,
17
-
50
.
Nicklas
R. B.
,
Staehly
C. A.
(
1967
).
Chromosome micromanipulation. I. The mechanics of chromosome attachment to the spindle
.
Chromosoma
21
,
1
-
16
.
Ogino
H.
,
McConnell
W. B.
,
Grainger
R. M.
(
2006
).
Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease
.
Mech. Dev.
123
,
103
-
113
.
Paine
P. L.
,
Feldherr
C. M.
(
1972
).
Nucleocytoplasmic exchange of macromolecules
.
Exp. Cell Res.
74
,
81
-
98
.
Qiu
S.
,
C. M.
,
Lane
T.
(
2005
).
A computational study of off-target effects of RNA interference
.
Nucleic Acid Res.
33
,
1834
-
1847
.
Taylor
D. L.
,
Wang
Y. L.
(
1978
).
Molecular cytochemistry: incorporation of fluorescently labeled actin into living cells
.
75
,
857
-
861
.
Waldner
C.
,
Sakamaki
K.
,
Ueno
N.
,
Turan
G.
,
Ryffel
G. U.
(
2006
).
Transgenic Xenopus laevis strain expressing cre recombinase in muscle cells
.
Dev. Dyn.
235
,
2220
-
2228
.
Zhang
C.
,
Carl
T. F.
,
Trudeau
E. D.
,
Simmet
T.
,
Klymkowsky
M. W.
(
2006
).
An NF-kappaB and slug regulatory loop active in early vertebrate mesoderm
.
PLoS ONE
1
,
e106
.