Microinjection of mRNAs and Oligonucleotides

Abstract

Microinjecting lineage tracers into a single blastomere in the normal, intact embryo identifies the repertoire of cell types derived from it. In order to reveal the full developmental potential of that blastomere or identify the mechanisms by which its fate is determined, one needs to modify its gene expression under controlled experimental conditions. One method by which this is easily accomplished in Xenopus is by microinjecting synthetic mRNAs or antisense oligonucleotides into an identified blastomere to target altered gene expression specifically to its lineage. Xenopus blastomeres are robust and tolerate pressure-driven microinjection up to a few hundred cells, and they efficiently translate exogenously supplied mRNAs. Targeted microinjections, described here, significantly reduce off-target effects of the mRNAs or oligonucleotides. Many types of constructs can be synthesized to provide specific information about gene function. For example, microinjecting mRNA encoding the wild-type gene in its normal expression domain or in an ectopic site tests whether it promotes or represses target genes or alters the formation of tissues of interest. Mutant forms of a gene transcript can illuminate the function of different domains of the encoded protein or show the developmental consequences of a mutation found in a human disease. mRNAs encoding dominant-negative forms of a protein can elicit a functional knockdown and thereby establish the necessity for that gene in a developmental process. Microinjecting antisense morpholino oligonucleotides (MOs) that are designed to block either endogenous mRNA translation or splicing is an effective method to reduce the levels of endogenous protein. MATERIALSIt is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol. RECIPES: Please see the end of this protocol for recipes indicated by <R>. Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes. ReagentsAgarose, 2% in 1× culture medium (if preparing agarose injection dishes in Step 1)Add 2 g of electrophoresis-grade agarose to 100 mL of 1× culture medium in a screw-cap glass bottle. Autoclave to dissolve agarose. This can be stored in the refrigerator, and microwaved to liquefy the agarose when it is next needed.

Nanomedicines to Deliver mRNA: State of the Art and Future Perspectives

“…This technique has shown efficient mRNA delivery for tumor antigen loading of DCs [81]; even in some studies, mRNA electroporation has been more efficient than DNA electroporation, with faster and more homogeneous protein expression in vivo [82]. The direct injection of mRNA into the target cell by the use of microneedles (namely, microinjection) [83,84], and gene gun, in which naked mRNA is pneumatically shot into the target cell, have also been used for mRNA transfection [85][86][87][88]. In in vivo applications, intravenously administered naked mRNA is rapidly degraded by ribonucleases and the innate immune system can be activated.…”

Using Xenopus to analyze neurocristopathies like Kabuki syndrome

“…Conversely, gain‐of‐function experiments can be performed by injecting RNA or DNA constructs. In addition, while targeted injections allow to dissect tissue‐specific functions (Moody, 2018), the use of chemical inhibitors as well as activators can serve to modulate signaling pathways at specific time points (Maj et al, 2016). Together with the technical means of rescue experiments, Xenopus is thereby ideally suited for the efficient and inexpensive analysis of candidate genes or genetic variants with a potential role in human disease (Bajpai et al, 2010; Devotta, Juraver‐Geslin, Gonzalez, Hong, & Saint‐Jeannet, 2016; Duncan & Khokha, 2016; Lienkamp, 2016; Martens et al, 2020; Ott et al, 2019; Pratt & Khakhalin, 2013; Schwenty‐Lara et al, 2020; Ufartes et al, 2018; Vivante et al, 2017).…”

“…Conversely, gain-of-function experiments can be performed by injecting RNA or DNA constructs. In addition, while targeted injections allow to dissect tissue-specific functions (Moody, 2018), the use of chemical inhibitors as well as activators can serve to modulate signaling pathways at specific time points (Maj et al, 2016).…”

The Many Faces of Xenopus: Xenopus laevis as a Model System to Study Wolf–Hirschhorn Syndrome

“…In order to characterize WHS-associated gene functions in relation to development and disease, Xenopus embryos can be injected with a variety of materials to manipulate gene expression, such as CRISPR/cas9 or morpholino oligonucleotides (MOs), in either the whole embryo or selected blastomeres (up to the 64-cell stage) (Mimoto and Christian, 2011; Tandon et al, 2012, 2017; Bestman and Cline, 2014; Bhattacharya et al, 2015; Wang et al, 2015; DeLay et al, 2016; Feehan et al, 2017; Moody, 2018a, b). As the lineage of individual cells has been well-documented, injections can be precisely targeted to specific tissues and organs that are affected in WHS, such as the heart, kidney, or brain.…”