The authors would like to thank all the members of the "Insect Genetics and Genomics" Laboratory, in particular to Lorenzo Ghiringhelli who has worked at developing, adapting and maintaining the rearing of the medfly over the past thirty years. Part of the representative results of this paper have been reprinted from N. Biotechnology, 25(1) by Scolari F. et al., Fluorescent sperm marking to improve the fight against the pest insect Ceratitis capitata (Wiedemann; Diptera: Tephritidae), 76-84, 2008, with permission from Elsevier (License number 3796240759880). This work received support from Cariplo-Regione Lombardia "IMPROVE" (FS).

1. Experimental Set-up
<ol>
	<li>Insectary requirements
	<ol>
		<li>Maintain all C. capitata life stages at 25 &#176;C, 65% humidity and 12/12 hr light/dark photoperiod.</li>
		<li>Place about 1,500-2,000 medfly pupae in a 6 L cage. Use a cage with a brass mesh on one side with holes small enough to stimulate oviposition42. Insert a sponge strip through a small opening in the cage&#39;s base to provide flies with water by means of capillary action. Use a mixture of yeast and sugar (1:10) as food source for the adults (Figure 1A). Increase the amount of yeast (up to 1:3, yeast:sugar) should the females fail to lay eggs.</li>
		<li>Place a plate filled with water below the cage to collect the eggs deposited through the mesh. As virgin females can oviposit, wait until the flies are at least 6-7 days old before collecting eggs for the microinjection experiments. This will ensure that the females will have mated and that the eggs will have been fertilized.</li>
		<li>Collect the eggs by filtering the water with a fine net strainer. Transfer the eggs from the strainer in a plastic box containing standard larval food (1.5 L H2O, 100 ml HCl 1%, 5 g broad-spectrum antimicrobial agent dissolved in 50 ml ethanol, 400 g sugar, 175 g demineralized brewer&#39;s yeast, 1 kg soft wheat bran) using a Pasteur pipette.</li>
		<li>Place each box in a transparent container closed with a net lid to allow air circulation and containing a layer of bran to favor pupation (Figure 1B).</li>
		<li>After about 10 days, check for 3rd instar larvae that emerge from the larval food and jump down into the bran layer for pupation.</li>
		<li>24 hr later, collect the pupae in a small cup using a soft brush and transfer them to a new adult cage. Adults normally emerge 10 days after pupation. The emerging fly uses the ptilinum, an inflatable pocket located on its head above the base of the antennae, to force off the end of the puparium. After emergence, the ptilinum permanently collapses back inside the head.</li>
	</ol>
	</li>
	<li>Preparation of 1 L food for microinjected larvae
	<ol>
		<li>Dissolve 2.5 g agar in 300 ml H2O.</li>
		<li>Warm 500 ml H2O on a hot stirring plate and dissolve 4 g Sodium Benzoate, 4.5 ml 37% HCl, 42 g yeast extract and 115 g dehydrated carrot powder.</li>
		<li>Add to the mixture a solution of 2.86 g broad-spectrum antimicrobial agent dissolved in 10 ml absolute ethanol.</li>
		<li>Distribute the medium in 15 cm round Petri dishes and allow to cool. If necessary, store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Slide preparation
	<ol>
		<li>Place a 2 cm strip of double-sided tape on a microscope slide (75 x 26 mm2) and mark the edges with a china marker, to prevent oil spread and the consequent desiccation of the injected embryos.</li>
	</ol>
	</li>
	<li>Nucleic Acid Preparation
	<ol>
		<li>Isolate plasmid DNA using commercially available kits34. Precipitate the plasmid using ethanol and re-suspend in injection buffer (0.1 mM phosphate buffer pH 7.4, 5 mM KCl) at the desired concentration.</li>
		<li>Purify in vitro synthetized long dsRNA using phenol-chloroform, precipitate using isopropanol and re-suspend in injection buffer43.</li>
	</ol>
	</li>
</ol>
2. Embryo Preparation
<ol>
	<li>Place a tray filled with water under a cage containing 6-7 day old adults.</li>
	<li>Allow the females to oviposit for 30 min and then collect the embryos by filtering the water using a fine strainer. Always keep the strainer in water to avoid desiccation of the embryos.</li>
	<li>Dechorionate the embryos by immerging the strainer for 5 sec in a commercial 50% bleach solution.</li>
	<li>Wash the embryos carefully by repeatedly immerging the strainer in clean ultrapure water (at least 4-5 times). Finally, place the strainer in clean ultrapure water. Use the embryos within maximum 2 hr. 
	NOTE: The timing of microinjection is related to the necessity to inject into pre-blastoderm embryos, during a phase of nuclear divisions prior to cellularization. This allows the injected DNA to be taken-up into the nuclei, specifically into the primordial germ cell nuclei that will originate the gametes. In Drosophila, cellularization at the pole cells begins at about 90 min after fertilization (at 25 &#176;C), with blastoderm formation occurring about 30 min later44, whereas in the medfly cellularization takes place between 9 and 12 hr45,46.</li>
	<li>Embryo orientation in rows
	<ol>
		<li>Using a fine paintbrush, collect about 50 embryos and place them on a disk of black filter paper soaked with water.</li>
		<li>Under a dissecting stereomicroscope, arrange the embryos in a row on the upper surface of the double-sided tape on the microscope slide using a fine paintbrush (000) (Figure 1C). 
		NOTE: Align the embryos all in the same orientation, as injection is performed in the posterior pole, where germ-line will originate; the posterior pole is opposite to the micropyle region.</li>
		<li>Cover the embryos with a layer of chlorotrifluoroethylene oil making sure that the oil does not spill over china marker borders (see 1.3). 
		NOTE: Before covering the embryos with oil, a further step can be performed: the embryos can be desiccated for a few minutes to reduce their liquid content and thus facilitate the entry of the injected DNA solution. This may help avoid embryo bursting during injection or excessive leakage of the injected solution, as a consequence of the high liquid pressure.</li>
	</ol>
	</li>
</ol>
3. Embryo Microinjection
<ol>
	<li>Fill a needle (1-2 &#181;l) with plasmid DNA or dsRNA solution (1-2 &#181;g/&#181;l) using a micropipette.</li>
	<li>Connect the needle to the microinjection apparatus, which controls the injection time (0.5 sec), the pressure (800 hPa) and the backpressure (150 hPa).</li>
	<li>Perform microinjection under a stereomicroscope, using a micromanipulator to control the movements of the needle (Figure 1D). Use the microscope stage to move the slide.
	<ol>
		<li>Place the slide on the stage of the scope.</li>
		<li>Insert the tip of the needle into the posterior pole of the embryo ( Figure 1E ).</li>
		<li>Inject the solution by activating the microinjection system with the pedal. The injected liquid induces an increase in internal pressure, resulting in a slight movement of the embryo.</li>
		<li>Release the pedal and immediately, but gently, remove the needle from the embryo.</li>
		<li>Observe a little cytoplasmic leakage at the injection site. 
		NOTE: To discriminate between potential lethal effects of DNA/dsRNA/siRNA and mortality due to the microinjection procedure itself, control experiments must be performed. These comprise the injection of buffer only, or, in the case of reverse genetics experiments, of a green fluorescent protein (GFP)-derived double-stranded RNA (dsRNA-GFP), or siRNA.</li>
	</ol>
	</li>
	<li>Following injection incubate the embryos at 25 &#176;C.</li>
</ol>
4. Post-injection Procedures
<ol>
	<li>Two days after injection, check the slides for hatched larvae (Figure 1F) under a stereomicroscope. Larvae can move in the oil but need to be transferred to larval food as soon as possible. For this reason, check the slides several times a day.</li>
	<li>Using a fine paintbrush (000), gently transfer the hatched larvae to the carrot-based larval food. Transfer up to a maximum of 200 larvae per each Petri dish.</li>
	<li>Incubate the larvae at 65% humidity (25 &#176;C). Ensure that the lid does not stick to the dish, impairing air exchange.</li>
	<li>Keep the Petri dishes with the lid mostly closed so that there is still air flow to the larvae. To avoid excessive drying, check the dishes daily and, if needed, add water to compensate for evaporation.</li>
	<li>Place the Petri dishes in a clear container closed by a net-lid and containing a layer of bran to favor pupation. As for routine insectary rearing, 3rd instar larvae jump out of the larval food and pupate in the bran.</li>
	<li>Collect the pupae and place them in a small cage for adult rearing and screening (this is Generation 0, G0). Rear the progeny on standard larval food.</li>
	<li>In the case of germ-line transformation experiments, check for possible transformed individuals in the next generation (G1), as injected individuals usually are chimeras. Transposition events, indeed, might have occurred in any of the nuclei of the embryonic syncytium, which will form either the germ-line or the soma.
	<ol>
		<li>Soon after emergence, anesthetize G1 individuals using CO2 and check for transformation events under an epifluorescence stereomicroscope. To screen for the presence of red fluorescence, use a DsRedwide filter (Ext. 546/12; Emm. 605/75), while to screen for green fluorescence use the EYFP filter (Ext. 500/20; Emm. 535/30).</li>
		<li>Place transformed individuals in a small cage with wild-type adults of the opposite sex and originate a new, initially heterozygous, strain.</li>
	</ol>
	</li>
</ol>

The authors declare that they have no competing financial interests.

Microinjection of nucleic acids in insect embryos is a universal technique that facilitates both the analysis of gene function and biotechnological applications.
The recent publication of the genome sequences from an increasing number of insect species leads to an urgent need for tools for the functional characterization of genes of yet unknown function. RNA-interference has proven to be one of the most valuable methods to infer molecular functions49 and embryo microinjection facili...

The Mediterranean fruit fly (medfly) Ceratitis capitata is a cosmopolitan species that extensively damages fruits and cultivated crops. It belongs to the Tephritidae family, which includes several pest species, such as those belonging to the genera Bactrocera and Anastrepha. The medfly is the most studied species of this family, and it has become a model not only for the study of insect invasions1, but also for optimizing pest management strategies2.
The medfly is a multivoltine species that can attack more than 300 species of wild and cultivated plants3,4. The damage is caused by both the adults and the larval stages: mated females pierce the surface of the fruit for oviposition, allowing microorganisms to affect their commercial quality, whereas the larvae feed on the fruit pulp. After three larval stages, larvae emerge from the host and pupate into the soil. Ceratitis capitata displays an almost worldwide distribution, including Africa, the Middle-East, Western Australia, Central and South America, Europe, and areas of the United States5.
The most common strategies to limit medfly infestations involve the use of insecticides (e.g., Malathion, Spinosad) and the environmentally-friendly Sterile Insect Technique (SIT)6. The latter approach involves the release into the wild of hundreds of thousands of males rendered sterile by exposure to ionizing irradiation. The mating of such sterilized males to wild females results in no progeny, causing a reduction in population size, eventually leading to eradication. Although SIT has proven effective in multiple campaigns worldwide, its major drawbacks include the high costs of rearing and sterilizing millions of insects to be released. Marking of released individuals is necessary to distinguish sterile from wild insects captured in the field during monitoring activities and it is currently achieved using fluorescent powders. These procedures are costly and have undesirable side-effects7.
In order to optimize and/or to develop more effective approaches for the control of this pest, medfly biology and genetics have been widely explored by numerous researchers worldwide. The availability of the medfly genome sequence8,9, will facilitate novel investigations on gene functions. RNA interference is a powerful tool for such studies and it can be achieved through the microinjection of dsRNA (double-stranded RNA) or siRNA (small interfering RNA). This technique has been used, for example, to demonstrate that the sex determination molecular cascade in C. capitata is only partially conserved with respect to that of Drosophila10.
The development of protocols to microinject medfly embryos allowed C. capitata to be the first non-Drosophilid fly species to be genetically modified. As its eggs are similar to those of Drosophila, both in terms of morphology and resistance to desiccation11, the protocol to deliver plasmid DNA into pre-blastoderm embryos first developed for D. melanogaster12,13 was initially adapted for use in C. capitata. These first experiments allowed medfly germ-line transformation based on the transposable element Minos11. Subsequently, the original system was modified14 using other transposon-based approaches. This is the case of&#160;piggyBac from the Lepidoptera Trichoplusia ni15. The protocol has since been further optimized and this has permitted the transformation of other tephritid species16-21 and also of many other Diptera22-31. All these systems rely on the use of a typical binary vector/helper plasmid transformation system: artificial, defective transposons containing desired genes are assembled into plasmid DNA and integrated into the genome of the insect by supplying the transposase enzyme32. A number of transgenic medfly lines have been generated, with multiple features including strains carrying a conditional dominant lethal gene that induces lethality, strains producing male-only progeny and thus not requiring additional sexing strategies, and strains with fluorescent sperm, which may enhance the accuracy of the SIT monitoring phase33-37. Although the release in the wild of transgenic organisms has occurred in pilot tests against mosquitoes only38,39, at least one company is evaluating a number of transgenic medfly strains for their use in the field40.
Embryo microinjection can also favor the development of new genome-editing tools, such as transcription activator-like effector nucleases (TALENs), clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated protein 9 nuclease (Cas9) and homing endonucleases genes (HEGs), which will enable novel evolutionary and developmental studies, as well as expanding the available biotechnological toolbox. Genome-editing approaches already allowed the generation of gene-drive systems in mosquitoes41, and their transfer to the medfly is imminent. Here we describe a universal protocol for microinjecting nucleic acids in medfly embryos that can be useful for all the above mentioned applications.

<table border="0" cellpadding="0" cellspacing="0" width="791">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1 x injection Buffer&#160;</td>
			<td style="width:107px;">Buffer</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">0.1 mM phosphate buffer pH 7.4, 5mM KCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Construct Plasmid</td>
			<td style="width:107px;">DNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Helper Plasmid</td>
			<td style="width:107px;">DNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dsRNA</td>
			<td style="width:107px;">RNA</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Phenol-Chloroform purified</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:216px;">Standard Larval food</td>
			<td style="width:107px;">Rearing Food&#160;</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">1.5 L H2O, 100 ml HCl 1%, 5 g broad-spectrum antimicrobial agent used in pharmaceutical products&#160; dissolved in 50 ml of ethanol, 400 g sugar, 175 g demineralized brewer&#8217;s yeast, 1 kg soft wheat bran</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Carrot Larval Food</td>
			<td style="width:107px;">Rearing food</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">2.5 g Agar, 4 g Sodium Benzoate, 4.5 ml 37% HCl, 42 g yeast extract, 115 g carrot powder, 2.86 g broad-spectrum antimicrobial agent , water to 1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult Food</td>
			<td style="width:107px;">Rearing food</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td>yeast extract and sugar (1:10)&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microscope slides</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td>Z692247</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injection needles&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Eppendorf</td>
			<td align="right">5242956000</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microloaders</td>
			<td style="width:107px;"></td>
			<td style="width:93px;">Eppendorf</td>
			<td align="right">5242956003</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Double slided tape</td>
			<td style="width:107px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:216px;">Whatman Black circle paper</td>
			<td style="width:107px;"></td>
			<td style="width:93px;"></td>
			<td></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:107px;">Generic reagent</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">Diluite 1:2 before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paintbrush (000)</td>
			<td style="width:107px;">Generic tool</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micromanipulator</td>
			<td style="width:107px;">Instrument</td>
			<td style="width:93px;">Narishige</td>
			<td style="width:119px;">MN-153</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Microinjector</td>
			<td style="width:107px;">Instrument</td>
			<td style="width:93px;">Eppendorf</td>
			<td style="width:119px;">Femtojet</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Adult cages</td>
			<td style="width:107px;">Generic tool</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Halocarbon oil 700</td>
			<td style="width:107px;">Reagent</td>
			<td style="width:93px;">Sigma-Aldrich</td>
			<td style="width:119px;">H8898</td>
			<td style="width:257px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ceratitis capitata</td>
			<td style="width:107px;">Animal</td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td style="width:257px;">The strain used is ISPRA</td>
		</tr>
	</tbody>
</table>

delivery of nucleic acids through embryo microinjection in the worldwide agricultural pest insect, ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. This is due to its reproductive behavior: females damage the external surface of fruits and vegetables when they lay eggs and the hatched larvae feed on their pulp. Wild C. capitata populations are traditionally controlled through insecticide spraying and/or eco-friendly approaches, the most successful being the Sterile Insect Technique (SIT). The SIT relies on mass-rearing, radiation-based sterilization and field release of males that retain their capacity to mate but are not able to generate fertile progeny. The advent and the subsequent rapid development of biotechnological tools, together with the availability of the medfly genome sequence, has greatly boosted our understanding of the biology of this species. This favored the proliferation of new strategies for genome manipulation, which can be applied to population control.
In this context, embryo microinjection plays a dual role in expanding the toolbox for medfly control. The ability to interfere with the function of genes that regulate key biological processes, indeed, expands our understanding of the molecular machinery underlying medfly invasiveness. Furthermore, the ability to achieve germ-line transformation facilitates the production of multiple transgenic strains that can be tested for future field applications in novel SIT settings. Indeed, genetic manipulation can be used to confer desirable traits that can, for example, be used to monitor sterile male performance in the field, or that can result in early life-stage lethality. Here we describe a method to microinject nucleic acids into medfly embryos to achieve these two main goals.

Here we report two applications of embryo microinjection directed at the functional characterization of a gene of interest (Case 1), and at the generation of transgenic strains (Case 2), respectively.
Delivery of dsRNA into embryos to unravel gene function.
The innexin-5 gene encodes a gap-junction that, in insects, is expressed specifically in the male and fe...

Watch this Scientific Journal Video about Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata at JoVE.com

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Mediterranean fruit fly (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) is a pest species with extremely high agricultural relevance. ...

delivery-nucleic-acids-through-embryo-microinjection-worldwide

Research

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Genetics

8.9K Views.  University of Pavia. The Mediterranean fruit fly (medfly) Ceratitis capitata (Diptera: Tephritidae) is a worldwide pest of agriculture. A deeper understanding of its biology is key to control medfly populations and thus reduce economic impact. Embryo microinjection is a fundamental tool allowing both germ-line transformation and reverse genetics studies in this species.

Video: Delivery of Nucleic Acids through Embryo Microinjection in the Worldwide Agricultural Pest Insect, Ceratitis capitata

The Terroir Concept Interpreted through Grape Berry Metabolomics and Transcriptomics

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to particular habitats and management practices. This article shows how certain terroir signatures can be detected in the berry metabolome and transcriptome of the grapevine cultivar Corvina using multivariate statistical analysis. The method first requires an appropriate sampling plan. In this case study, a specific clone of the Corvina cultivar was selected to minimize genetic differences, and samples were collected from seven vineyards representing three different macro-zones during three different growing seasons. An untargeted LC-MS metabolomics approach is recommended due to its high sensitivity, accompanied by efficient data processing using MZmine software and a metabolite identification strategy based on fragmentation tree analysis. Comprehensive transcriptome analysis can be achieved using microarrays containing probes covering ~99% of all predicted grapevine genes, allowing the simultaneous analysis of all differentially expressed genes in the context of different terroirs. Finally, multivariate data analysis based on projection methods can be used to overcome the strong vintage-specific effect, allowing the metabolomics and transcriptomics data to be integrated and analyzed in detail to identify informative correlations.

This article describes the application of untargeted metabolomics, transcriptomics and multivariate statistical analysis to grape berry transcripts and metabolites in order to gain insight into the terroir concept, i.e., the impact of the environment on berry quality traits.

Terroir refers to the combination of environmental factors that affect the characteristics of crops such as grapevine (Vitis vinifera) according to ...

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Sequence-specific and Selective Recognition of Double-stranded RNAs over Single-stranded RNAs by Chemically Modified Peptide Nucleic Acids

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands for the recognition of RNA sequence and structure. Chemically modified Peptide Nucleic Acid (PNA) oligomers have been recently developed that can recognize RNA duplexes in a sequence-specific manner. PNAs are chemically stable with a neutral peptide-like backbone. PNAs can be synthesized relatively easily by the manual Boc-chemistry solid-phase peptide synthesis method. PNAs are purified by reverse-phase HPLC, followed by molecular weight characterization by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). Non-denaturing polyacrylamide gel electrophoresis (PAGE) technique facilitates the imaging of the triplex formation, because carefully designed free RNA duplex constructs and PNA bound triplexes often show different migration rates. Non-denaturing PAGE with ethidium bromide post staining is often an easy and informative technique for characterizing the binding affinities and specificities of PNA oligomers. Typically, multiple RNA hairpins or duplexes with single base pair mutations can be used to characterize PNA binding properties, such as binding affinities and specificities. 2-Aminopurine is an isomer of adenine (6-aminopurine); the 2-aminopurine fluorescence intensity is sensitive to local structural environment changes, and is suitable for the monitoring of triplex formation with the 2-aminopurine residue incorporated near the PNA binding site. 2-Aminopurine fluorescence titration can also be used to confirm the binding selectivity of modified PNAs towards targeted double-stranded RNAs (dsRNAs) over single-stranded RNAs (ssRNAs). UV-absorbance-detected thermal melting experiments allow the measurement of the thermal stability of PNA-RNA duplexes and PNA&#183;RNA2 triplexes. Here, we describe the synthesis and purification of PNA oligomers incorporating modified residues, and describe biochemical and biophysical methods for characterization of the recognition of RNA duplexes by the modified PNAs.

We report the protocols for the synthesis and purification of Peptide Nucleic Acid (PNA) oligomers incorporating modified residues. The biochemical and biophysical methods for the characterization of the recognition of RNA duplexes by the modified PNAs are described.

RNAs are emerging as important biomarkers and therapeutic targets. Thus, there is great potential in developing chemical probes and therapeutic ligands ...

Transient Expression of Foreign Genes in Insect Cells (sf9) for Protein Functional Assay

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell culture system. This system was developed to express foreign genes under the control of the baculoviral promoter in transient expression plasmids. Furthermore, this system can be applied to a functional assay of either the baculovirus itself or foreign proteins. The most widely and commercially available transient gene expression system is developed based on the immediate-early gene (IE) promoter of Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV). However, a low expression level of foreign genes in insect cells was observed. Therefore, a transient gene expression system was constructed for improving protein expression. In this system, recombinant plasmids were constructed to contain the target sequence under the control of the Drosophila heat shock 70 (Dhsp70) promoter. This protocol presents the application of this heat shock-based pDHsp/V5-His (V5 epitope with 6 histidine)/Spodoptera frugiperda cell (sf9 cell) system; this system is available not only for gene expression but also for evaluating the anti-apoptotic activity of candidate proteins in insect cells. Furthermore, this system can be either transfected with one recombinant plasmid or co-transfected two potentially functionally antagonistic recombinant plasmids in insect cells. The protocol demonstrates the efficiency of this system and provides a practical case of this technique.

Transient Expression of Foreign Genes in Insect Cells sf9 for Protein Functional Assay

This protocol describes a heat shock-induced protein expression system (pDHsp/V5-His/sf9 cell system), which can be used for either expressing foreign proteins or evaluating the anti-apoptotic activity of potential foreign proteins and their truncated amino acids in insect cells.

The transient gene expression system is one of the most important technologies for performing protein functional analysis in the baculovirus in vitro cell ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species alike. This technology can be used for a variety of genome editing approaches, from gene knockouts and knockins to more specific changes like the introduction of a few nucleotides at a targeted location. Genome editing can be used for a multitude of applications, including the partial functional characterization of genes, the production of transgenic organisms and the development of diagnostic tools. Compared to previously available gene editing strategies, the CRISPR-Cas9 system has been shown to be easy to establish in new species and boasts high efficiency and specificity. The primary reason for this is that the editing tool uses an RNA molecule to target the gene or sequence of interest, making target molecule design straightforward, given that standard base pairing rules can be exploited. Similar to other genome editing systems, CRISPR-Cas9-based methods also require efficient and effective transformation protocols as well as access to good quality sequence data for the design of the targeting RNA and DNA molecules. Since the introduction of this system in 2013, it has been used to genetically engineer a variety of model species, including Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster and Mus musculus. Subsequently, researchers working on non-model species have taken advantage of the system and used it for the study of genes involved in processes as diverse as secondary metabolism in fungi, nematode growth and disease resistance in plants, among many others. This protocol detailed below describes the use of the CRISPR-Cas9 genome editing protocol for the truncation of a gene involved in the sexual cycle of Huntiella omanensis, a filamentous ascomycete fungus belonging to the Ceratocystidaceae family.

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete fungus.

The CRISPR-Cas9 genome editing system is a molecular tool that can be used to introduce precise changes into the genomes of model and non-model species ...