We appreciate the assistance of the NHGRI mouse facility staff, the NCI Molecular Pathology Lab, and especially Andrew Warner. R.J.C. is supported by the Intramural Research Program of the NHGRI through 1ZIAHG200318-16 and this work was partially funded by the National Center for Advancing Translational Sciences (NCATS). Figure 2C was created with BioRender.

This protocol and the accompanying video are for the retrobulbar injection space of postnatal, juvenile, and runted adult mice by retrobulbar sinus injection; the protocol has been approved by the National Human Genome Research Institute&#39;s Institutional Animal Care and Use Committee (ACUC) under protocol number G-03-4. Other institutions may have different requirements and restrictions, and this protocol may need to be modified for approval in your institute. Obtain approval from your institution&#39;s ACUC before performing this or any other animal procedure.
1. Pre-injection preparation
<ol>
	<li>Dilute the AAV to the desired injection volume and concentration with sterile phosphate-buffered saline (PBS) in a sterile 1.5 mL microcentrifuge tube for each injection. Add an extra 50% volume for each injection to allow for accurate filling of the single-use sterile syringe. 
	NOTE: Here we are diluting an AAV8-CAG-eGFP reporter to deliver a dose of 1 &#215; 1013viral genomes per kg of body mass (vg/kg) in a volume of 50 &#956;L. The amount of AAV in the 50 &#956;L to be injected is calculated using the weight of the animal at the time of injection. In this video, AAV is being injected via the retrobulbar route to replicate systemic delivery in humans. Other gene therapy vectors (i.e., lentivirus, adenovirus), RNA therapies, and small molecules can be delivered systemically using this procedure.</li>
	<li>Ensure that the table-top laboratory animal anesthesia system (LAAS) is properly set up and functioning correctly in accordance with the manufacturer&#39;s instructions. 
	NOTE: If an alternative anesthesia method is used, ensure that anethesia is prepared prior to beginning injections. The retrobulbar injection procedure should be compatible with most anesthesias (e.g., an injectable chemical restraint like ketamine and xylazine).</li>
	<li>Prior to filling the single-use sterile syringe (here, an insulin syringe, 31 G, 8 mm long, 3/10 mL capacity), move the plunger up and down several times to ensure the plunger can be depressed smoothly. Then, fill the syringe to the desired volume, ensuring there are no air bubbles. 
	NOTE: Volumes up to 150 &#181;L can be injected; our lab typically injects a volume of 50 &#181;L.</li>
</ol>
2. Mouse sedation via administration of isoflurane gas with a laboratory animal anesthesia system (LAAS)
<ol>
	<li>Ensure that gas is only flowing to the induction chamber. Close the two-way stopcock to the non-rebreathing (NRB) circuit.</li>
	<li>Turn on the green oxygen flow knob on the front of the flow meter so that there is a flow rate of 1 L/min.</li>
	<li>Turn isoflurane to &#8804;4% by depressing the lever at the top of the vaporizer and turning the dial to the desired concentration.</li>
	<li>Place the mouse in the clear induction chamber. Watch the animal&#39;s breathing and movement carefully. Once the animal is recumbent, turn the vaporizer knob down to 2-2.5% of isoflurane.</li>
	<li>Open the two-way stopcock to the NRB circuit attached to the face mask and close the gas flow to the induction box.</li>
	<li>Remove the animal and place it in the NRB circuit&#39;s face mask.</li>
	<li>Turn down the isoflurane concentration setting to 1.5%-1.75%, as determined by reaction to stimuli (e.g., toe-pinch or paw-squeeze).</li>
	<li>Always continuously monitor the mouse&#39;s breathing and mucous membrane color (if possible). If the animal&#39;s breathing becomes labored or the mucous membrane color is not pink, turn down the anesthetic concentration.</li>
	<li>Keep the animal warm throughout the entire procedure. Use a handwarmer wrapped in a paper towel that is placed underneath the underpad and located directly underneath the mouse.</li>
	<li>Turn off the oxygen and the vaporizer after completion of the procedure.</li>
</ol>
3. Injection of the animal
<ol>
	<li>If right-handed, inject the mouse&#39;s right eye and position the mouse on its left side with the snout pointing towards the right hand. If left-handed, inject the mouse&#39;s left eye and position the mouse on its right side with the snout pointing towards the left hand.</li>
	<li>Apply one or two drops of ophthalmic anesthetic to the eyeball, which is to be injected. Then, remove any excess ophthalmic anesthetic solution using a sterile absorbent gauze pad. Apply gentle pressure with fingertips to the skin dorsal and ventral to the eye to partially protrude the mouse&#39;s eyeball from the socket (Figure 2A,B). 
	NOTE: Be careful not to apply excessive pressure to surrounding cervical vessels when protruding the eye as this will impede blood flow and injection. In addition, applying pressure to the trachea could prevent the mouse from breathing. Ensure the mouse can breathe throughout the procedure.</li>
	<li>Keep the needle in a bevel-down position at an angle of approximately 30&#176; and placed into the medial canthus (Figure 2C). 
	NOTE: The depth of the needle placement to reach the retrobulbar sinus will vary based on the size of the animal. Having the needle in the bevel-down position during the injection decreases the risk of ocular damage. Be careful not to place the needle too deep and puncture the eye socket. The injection should take less than a minute.</li>
	<li>Slowly and smoothly apply pressure to the syringe plunger to deliver the injectate. This will reduce the chance of extravasation.</li>
	<li>Remove the needle slowly and smoothly.</li>
	<li>Remove the face mask from the mouse to allow for recovery from anesthesia.</li>
</ol>
4. Post injection
<ol>
	<li>Turn off the oxygen and the vaporizer after completion of the procedure.</li>
	<li>Use sterile gauze to remove blood if residual bleeding occurs.</li>
	<li>Ensure the mouse is in a warm area (approximately 37 &#176;C), but not excessively hot, to prevent hypothermia during recovery from anesthesia.</li>
	<li>Observe the mouse in isolation until completely recovered before returning the mouse to the cage and rack. 
	NOTE: Isolating the mouse during recovery prevents cage mates from injuring the sedated mouse during recovery.</li>
</ol>

Effective Systemic Delivery in Mice Using Retrobulbar Injection Technique

<ol>
	<li>Perlman, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+human+disease:+An+evolutionary+perspective.">Mouse models of human disease: An evolutionary perspective.</a> Evol Med Public Health. 2016 (1), 170-176 (2016).</li><li>Samelson-Jones, B. J., George, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adeno-associated+virus+gene+therapy+for+hemophilia.">Adeno-associated virus gene therapy for hemophilia.</a> Annu Rev Med. 74, 231-247 (2023).</li><li>Yardeni, T., Eckhaus, M., Morris, H. D., Huizing, M., Hoogstraten-Miller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrobulbar+injections+in+mice.">Retrobulbar injections in mice.</a> Lab Anim (NY). 40 (5), 155-160 (2011).</li><li>Chandler, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+gene+therapy+for+methylmalonic+acidemia+using+the+novel+adeno-associated+viral+vector+44.9.">Systemic gene therapy for methylmalonic acidemia using the novel adeno-associated viral vector 44.9.</a> Mol Ther Methods Clin Dev. 27, 61-72 (2022).</li><li>Chandler, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+aav9+gene+therapy+improves+the+lifespan+of+mice+with+niemann-pick+disease,+type+c1.">Systemic aav9 gene therapy improves the lifespan of mice with niemann-pick disease, type c1.</a> Hum Mol Genet. 26 (1), 52-64 (2017).</li><li>Venturoni, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+advantage+of+corrected+hepatocytes+in+a+juvenile+model+of+methylmalonic+acidemia+following+liver+directed+adeno-associated+viral+mediated+nuclease-free+genome+editing.">Growth advantage of corrected hepatocytes in a juvenile model of methylmalonic acidemia following liver directed adeno-associated viral mediated nuclease-free genome editing.</a> Mol Genet Metab. 137 (1-2), 1-8 (2022).</li><li>Ilyinskii, P. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immtor+nanoparticles+enhance+aav+transgene+expression+after+initial+and+repeat+dosing+in+a+mouse+model+of+methylmalonic+acidemia.">Immtor nanoparticles enhance aav transgene expression after initial and repeat dosing in a mouse model of methylmalonic acidemia.</a> Mol Ther Methods Clin Dev. 22, 279-292 (2021).</li><li>Davidson, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+systemic+aav+gene+therapy+with+a+neurotrophic+capsid+in+niemann-pick+disease+type+c1+mice.">Improved systemic aav gene therapy with a neurotrophic capsid in niemann-pick disease type c1 mice.</a> Life Sci Alliance. 4 (10), e202101040 (2021).</li><li>Chandler, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+gene+therapy+using+an+aav44.9+vector+rescues+a+neonatal+lethal+mouse+model+of+propionic+acidemia.">Systemic gene therapy using an aav44.9 vector rescues a neonatal lethal mouse model of propionic acidemia.</a> Mol Ther Methods Clin Dev. 30, 181-190 (2023).</li><li>Rocha-Ferreira, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+neonatal+rodent+model+of+retroorbital+vein+injection.">A neonatal rodent model of retroorbital vein injection.</a> J Vis Exp. (204), e65386 (2024).</li><li>Bohnert, B. N., Artunc, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+nephrotic+syndrome+in+mice+by+retrobulbar+injection+of+doxorubicin+and+prevention+of+volume+retention+by+sustained+release+aprotinin.">Induction of nephrotic syndrome in mice by retrobulbar injection of doxorubicin and prevention of volume retention by sustained release aprotinin.</a> J Vis Exp. (135), e57642 (2018).</li><li>Bohnert, B. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrobulbar+sinus+injection+of+doxorubicin+is+more+efficient+than+lateral+tail+vein+injection+at+inducing+experimental+nephrotic+syndrome+in+mice:+A+pilot+study.">Retrobulbar sinus injection of doxorubicin is more efficient than lateral tail vein injection at inducing experimental nephrotic syndrome in mice: A pilot study.</a> Lab Anim. 53 (6), 564-576 (2019).</li><li>Meijer, M. K., Spruijt, B. M., Van Zutphen, L. F., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+restraint+and+injection+methods+on+heart+rate+and+body+temperature+in+mice.">Effect of restraint and injection methods on heart rate and body temperature in mice.</a> Lab Anim. 40 (4), 382-391 (2006).</li><li>Nohara, M., Tohei, A., Sato, T., Amao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+response+to+restraint+stress+by+salivary+corticosterone+levels+in+adult+male+mice.">Evaluation of response to restraint stress by salivary corticosterone levels in adult male mice.</a> J Vet Med Sci. 78 (5), 775-780 (2016).</li><li>Schoch, A., Thorey, I. S., Engert, J., Winter, G., Emrich, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+routes+of+antibody+administration+in+pharmacokinetic+studies.">Comparison of the lateral tail vein and the retro-orbital venous sinus routes of antibody administration in pharmacokinetic studies.</a> Lab Anim (NY). 43 (3), 95-99 (2014).</li><li>Steel, C. D., Stephens, A. L., Hahto, S. M., Singletary, S. J., Ciavarra, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+lateral+tail+vein+and+the+retro-orbital+venous+sinus+as+routes+of+intravenous+drug+delivery+in+a+transgenic+mouse+model.">Comparison of the lateral tail vein and the retro-orbital venous sinus as routes of intravenous drug delivery in a transgenic mouse model.</a> Lab Anim (NY). 37 (1), 26-32 (2008).</li><li>Zincarelli, C., Soltys, S., Rengo, G., Rabinowitz, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+aav+serotypes+1-9+mediated+gene+expression+and+tropism+in+mice+after+systemic+injection.">Analysis of aav serotypes 1-9 mediated gene expression and tropism in mice after systemic injection.</a> Mol Ther. 16 (6), 1073-1080 (2008).</li></ol>

The authors have nothing relevant to this publication to disclose.

While retrobulbar injection is a reliable method to deliver small molecules, proteins, and genomic therapies, practicing the technique with a dye is necessary to ensure that reliable and replicable systemic delivery is achieved. The use of a dye is highly recommended for practicing retrobulbar injections in mice before using this delivery route in experiments. Dyes can be visually checked in the mouse tissues to ensure consistent systemic delivery.
In our demonstration of the retrobulbar injection technique, isoflurane gas was used to anesthetize the mice prior to the procedure. Other forms of anesthesia can be used prior to the procedure, but it is important to ensure that the mouse does not recover from the sedation before the injection is completed. Fortunately, the actual injection usually takes less than a minute and the time for which the mouse needs to be fully anesthetized is brief. The mouse should be completely sedated during the injection, and anesthesia should be readministered if the mouse becomes conscious prior to the injection. Since there are risks associated with using anesthesia, the duration of time the mouse is sedated should be minimized. We have not had issues using isoflurane to sedate small sick mice with methylmalonic and propionic acidemia. However, some mouse models may be more sensitive to sedation and certain anesthesia. This potential problem should be considered prior to attempting to use sedation in a study. Lastly, the use of sedation in combination with the retrobulbar injection greatly reduces the apparent distress the mouse exhibits during the injection process in comparison to TVI where sedation is not commonly used.
We have not observed any postinjection-related issues, although infection is a potential risk with any injection. To reduce the chance of infection a sterile single-use disposable syringe and sterile PBS to dilute the purified AAV are used. All mice in our animal facility are checked daily for signs of potential health issues and receive veterinary care to address any health issues when warranted.
The alternative to retrobulbar sinus injection and the more widely used method of systemic delivery to juvenile and adult mice is TVI. TVI and retrobulbar sinus injection result in a similar biodistribution in the case of small molecules and antibodies, and by extrapolation, the same would be expected for viral vectors15,16. However, no examples comparing the systemic delivery of gene therapy vectors by TVI and retrobulbar sinus injection could be found in the literature. In our opinion, retrobulbar sinus injections are easier to perform in mice with a decreased growth phenotype and/or early lethality.
TVI is often considered to be more analogous to systemic delivery in humans, despite humans having a retrobulbar sinus but not having a tail. In one aspect, the retrobulbar sinus injection is like human systemic delivery in that the injectant enters the upper venous system the same as if an injectant was delivered to a human by a peripherally inserted central catheter (PICC Line) or an intravenous catheter placed in the arm. Conversely, the injectant enters the lower venous system of a mouse after tail vein injection. Unfortunately, neither one of these methods exactly replicates the method(s) used for systemic delivery in humans, but both are effective methods of systemic delivery in mice.

Murine models of genetic diseases are commonly used to demonstrate the efficacy of small molecule, genetic, and cell therapies1. In mice, the most widely used method to replicate systemic delivery to humans is the tail vein injection (TVI), which is typically performed in adult mice at approximately 6-8 weeks of age to ensure the vein is large enough to access. TVI has been successfully used in numerous preclinical proof-of-principle studies of genetic diseases, such as hemophilia, that have supported human clinical trials for gene therapy2. However, many murine models of genetic disease have growth and/or early lethality phenotypes, which prevent them from reaching the age or size of an adult mouse (Figure 1). Treating such mice via TVI can be extremely difficult, if not impossible, depending on the age of lethality and/or the maximum size the animals can achieve.
In contrast, systemic delivery of a therapeutic agent by retrobulbar (frequently and incorrectly referred to as retro-orbital) sinus injection can be done quite readily in mice regardless of age or size3. Retrobulbar injections of adeno-associated virus (AAV) have been used successfully in young growth-retarded murine models of genetic disease, such as methylmalonic acidemia (MMA) and Niemann-Pick type C disease4,5,6,7,8. (This procedure can also be used to inject neonates3,4,9,10; however, this technique is not detailed in this protocol or the accompanying video.) Even highly toxic substances like doxorubicin can be delivered safely by retrobulbar injection11,12. Unlike TVI, mice are anesthetized during retrobulbar sinus injections, which makes the procedure less stressful on the mouse and easier on the operator who does not have to physically restrain the mouse13,14. An additional concern is that TVI frequently use a heat lamp to dilate the tail vein, which could potentially cause dehydration in young mice and could be problematic in murine models of genetic disease that are more suspectable to heat-related stress. Another problem that can arise when using TVI is that the tail vein can be particularly challenging to visualize on highly pigmented mice. However, like TVI, retrobulbar sinus injections result in a wide systemic biodistribution15,16.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AAV8-CAG-eGFP</td>
			<td>Univ. Penn. Vector Core</td>
			<td>Special order</td>
			<td>alternative sources of AAV reporters and alternative AAV reporters are available</td>
		</tr>
		<tr>
			<td>Barrier (Filter) Tips, 20 &#956;L size</td>
			<td>ThermoFisher</td>
			<td>AM12645</td>
			<td>for diluting AAV to disire injection volume and concentration</td>
		</tr>
		<tr>
			<td>Barrier (Filter) Tips, 100 &#956;L size (sterile)</td>
			<td>ThermoFisher</td>
			<td>AM12648</td>
			<td>for diluting AAV to disire injection volume and concentration</td>
		</tr>
		<tr>
			<td>Dual Prodedure Circuit&#160;</td>
			<td>VetEquip</td>
			<td>921400</td>
			<td>alternative anesthesia method can be used</td>
		</tr>
		<tr>
			<td>Gilsen PIPETTEMAN Classic P20 pipette</td>
			<td>Gilson</td>
			<td>F123600</td>
			<td>for diluting AAV to disire injection volume and concentration</td>
		</tr>
		<tr>
			<td>Gilsen PIPETTEMAN Classic&#160; P100 pipette</td>
			<td>Gilson</td>
			<td>F123615</td>
			<td>for diluting AAV to disire injection volume and concentration</td>
		</tr>
		<tr>
			<td>Hand warmers (HOTHANDS)</td>
			<td>ULINE</td>
			<td>S-1497B</td>
			<td>to keep mouse warm while anesthesized</td>
		</tr>
		<tr>
			<td>Insulin syringes, 31 G,&#160; 8 mm length, 3/10 mL capacity</td>
			<td>Becton Dickson</td>
			<td>328438</td>
			<td>used in video; one syringe per injection</td>
		</tr>
		<tr>
			<td>Isoflurane (Fluriso)</td>
			<td>VETONE</td>
			<td>502017</td>
			<td>alternative anesthesia can be used</td>
		</tr>
		<tr>
			<td>Medline Protection Plus Disposable Underpads</td>
			<td>ThermoFisher</td>
			<td>23-666-062</td>
			<td>to place mouse on durring injection</td>
		</tr>
		<tr>
			<td>Oak Ridge Phlebotomy Sharps Container With Transparent Lid</td>
			<td>ThermoFisher</td>
			<td>22-730-434</td>
			<td>for needle disposal</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td>To dilute AAV to desired concencentration and volume</td>
		</tr>
		<tr>
			<td>Snap Cap Low Retention Microcentrifuge Tubes (sterile)</td>
			<td>ThermoFisher</td>
			<td>3451</td>
			<td>for diluting AAV to disire injection volume and conentration</td>
		</tr>
		<tr>
			<td>Sterile gauze sponge 4&#34;x&#34;4</td>
			<td>Covidien</td>
			<td>3033</td>
			<td></td>
		</tr>
		<tr>
			<td>Table-Top laboratory animal anesthesia system (LAAS)</td>
			<td>VetEquip</td>
			<td>901806</td>
			<td>alternative anesthesia method can be used</td>
		</tr>
		<tr>
			<td>Trecaine Hydrochloride Ophthalmic (0.5%)</td>
			<td>Ocenanside Pharmaceuticals</td>
			<td>AK102D5DS</td>
			<td>local&#160; anesthetic</td>
		</tr>
		<tr>
			<td>Tuberculin syringe with a 27.0 G (or smaller)</td>
			<td>Any</td>
			<td>N/A</td>
			<td>alternative to insulin syringe used in video</td>
		</tr>
		<tr>
			<td>VetEquip&#8217;s User Guide and Operating Manual for table top and mobile Laboratory Animal Anesthesia System.&#160;</td>
			<td>VetEquip</td>
			<td>chrome-extension://efaidnbmnnnibpcajpcglclefindmkaj/https://www.vetequip.com/pdfs/LAAS%20Manual.pdf</td>
			<td>link to users guide and manual</td>
		</tr>
	</tbody>
</table>

systemic treatment for postnatal, juvenile, and runted adult mice by retrobulbar sinus injection

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for systemic delivery with fewer limitations. First, tail vein injections (TVIs) are limited to adult mice where the size of the tail vein is suitable for access. Being restricted to treating adult mice can be problematic when dealing with mouse models that do not survive to adulthood. Second, TVIs are not feasible for mouse models with growth retardation phenotypes where the mice never achieve the size of adult wildtype mice. Therefore, retrobulbar injections can be successfully used to treat both young and small adult mice. Lastly, retrobulbar injections are performed under anesthesia, which is less stressful on the mice than TVIs that are commonly performed without anesthesia. This article presents a protocol and detailed instructions for retrobulbar injections that can be used for systemic delivery to small and young mice.

Retrobulbar sinus injection has been used successfully to systemically deliver small molecules, antibodies, and adeno-associated virus (AAV)4,5,9,15,16. In Figure 3, the liver of a PBS (vehicle)-treated mouse and an AAV8-treated mouse liver are shown as an example of AAV injection and expression following a retrobulbar injection. AAV8, like many naturally occurring AAV vectors, is liver trophic. Therefore, substantial liver transduction is expected in a mouse that received a systemic dose of 5 &#215; 1012vg/kg17. The large number of hepatocytes expressing methylmalonyl-CoA mutase (MMUT)&#160;RNA seen in Figure 3, which is being expressed by the AAV transgene, indicates a successful retroorbital injection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66809/66809fig01.jpg" /> 
Figure 1: A growth retarded mouse with propionic acidemia. This is an example of the extreme growth retardation that can occur in murine models of genetic disease. <a href="https://www.jove.com/files/ftp_upload/66809/66809fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66809/66809fig02.jpg" /> 
Figure 2: Images and diagram of retrobulbar sinus injection. (A) Image of finger placement on fur to protrude eyeball (indicated by white arrow). (B) Image of eyeball (indicated by white arrow) protrusion after application of downward pressure on the fur prior to needle placement and injection. (C)&#160;Diagram of needle bevel orientation (bevel down relative to eyeball), needle angle (30&#176;), and retrobulbar sinus needle placement. <a href="https://www.jove.com/files/ftp_upload/66809/66809fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66809/66809fig03.jpg" /> 
Figure 3: RNA in situ hybridization after retrobulbar sinus Injection with AAV8. Images of (A)&#160;10x vehicle-treated liver, (B) 10x AAV8-treated liver, (C) 20x vehicle-treated liver, and (D)&#160;20x AAV8-treated liver stained for MMUT RNA. Mice with methylmalonic acidemia were treated with a dose of 5 &#215; 1012 vg/kg of AAV8-LPS-MMUT or a vehicle control (PBS) at 1 month of age. Liver tissue was collected 1 month post treatment. MMUT RNA stained brown (black arrows indicate areas of positive staining). Liver counterstained with hematoxylin. Scale bars = 100 &#181;m 10x for images, 50 &#181;m for 20x images (B). Abbreviations: AAV = adeno-associated virus; LPS = Liver-specific promoter; MMUT = methylmalonyl-CoA mutase. <a href="https://www.jove.com/files/ftp_upload/66809/66809fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Read this Scientific Article Protocol about Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection at JoVE.com

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 &#181;L for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for ...

systemic-treatment-for-postnatal-juvenile-runted-adult-mice

Research

JoVE Journal

Genetics

398 Views.  National Institutes of Health. This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 µL for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

Video: Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly useful. Hydrodynamic tail vein injection of transposon-based constructs is an efficient method for genetic manipulation of hepatocytes in adult mice. In addition to constitutive transgene expression, this system can be used for more advanced applications, such as shRNA-mediated gene knock-down, implication of the CRISPR/Cas9 system to induce gene mutations, or inducible systems. Here, the combination of constitutive CreER expression together with inducible expression of a transgene or miR-shRNA of choice is presented as an example of this technique. We cover the multi-step procedure starting from the preparation of sleeping beauty-transposon constructs, to the injection and treatment of mice, and the preparation of liver tissue for analysis by immunostaining. The system presented is a reliable and efficient approach to achieve complex genetic manipulations in hepatocytes. It is specifically useful in combination with Cre/loxP-based mouse strains and can be applied to a variety of models in the research of liver disease.

Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection

Hydrodynamic tail vein injection of transposon-based integration vectors enables stable transfection of murine hepatocytes in vivo. Here, we present a practical protocol for transfection systems that enables the long-term constitutive expression of a single transgene or combined constitutive and doxycycline-inducible expression of a transgene or miR-shRNA in the liver.

In research models of liver cancer, regeneration, inflammation, and fibrosis, flexible systems for in vivo gene expression and silencing are highly ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the only known non-legume lineage able to establish a nitrogen-fixing nodule symbiosis with rhizobium. Comparative studies between legumes and P. andersonii could provide valuable insight into the genetic networks underlying root nodule formation. To facilitate comparative studies, we recently sequenced the P. andersonii genome and established Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing. Here, we provide a detailed description of the transformation and genome editing procedures developed for P. andersonii. In addition, we describe procedures for the seed germination and characterization of symbiotic phenotypes. Using this protocol, stable transgenic mutant lines can be generated in a period of 2-3 months. Vegetative in vitro propagation of T0 transgenic lines allows phenotyping experiments to be initiated at 4 months after A. tumefaciens co-cultivation. Therefore, this protocol takes only marginally longer than the transient Agrobacterium rhizogenes-based root transformation method available for P. andersonii, though offers several clear advantages. Together, the procedures described here permit P. andersonii to be used as a research model for studies aimed at understanding symbiotic associations as well as potentially other aspects of the biology of this tropical tree.

Transforming, Genome Editing and Phenotyping the Nitrogen-fixing Tropical Cannabaceae Tree Parasponia andersonii

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae) and can form nitrogen-fixing root nodules in association with the rhizobium. Here, we describe a detailed protocol for reverse genetic analyses in P. andersonii based on Agrobacterium tumefaciens-mediated stable transformation and CRISPR/Cas9-based genome editing.

Parasponia andersonii is a fast-growing tropical tree that belongs to the Cannabis family (Cannabaceae). Together with 4 additional species, it forms the ...

Analyzing Satellite Cell Function During Skeletal Muscle Regeneration by Cardiotoxin Injury and Injection of Self-delivering siRNA In Vivo

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite cells. Satellite cells are characterized by the expression of the transcription factor Pax7 and their location underneath the basal lamina in the resting skeletal muscle. Upon injury, satellite cells get activated, undergo self-renewal or differentiation to either form new myofibers or to fuse with damaged ones. The functionality of satellite cells in vivo can be investigated using a cardiotoxin based injury model of skeletal muscle. To study the function of one gene during the regeneration of skeletal muscle, transgenic mouse models are mostly used. Here, we present an alternative method to transgenic mice, to investigate the gene function in satellite cells during regeneration, e.g., in cases where transgenic mice are not available. We combine the cardiotoxin mediated injury of a specific skeletal muscle with the injection of a self-delivering siRNA into the regenerating muscle which is then taken up by satellite cells among other cells. Thereby, we provide a method to analyze gene function in satellite cells during regeneration under physiological conditions without the need for transgenic mice.

We describe an in vivo method to investigate the functional loss of a specific gene in satellite cells by using a combination of cardiotoxin mediated injury of skeletal muscle and the injection of a self-delivering siRNA.

Skeletal muscle possesses an enormous capacity to regenerate after injury. This process is mainly driven by muscle stem cells, also termed satellite ...

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate regulation of these processes. A strategy called CLIP-seq (cross-linking and immunoprecipitation) has been developed to capture endogenous protein-RNA interactions with UV cross-linking followed by immunoprecipitation. Despite the wide use of conventional CLIP-seq method in RBP study, the CLIP method is limited by the availability of high-quality antibodies, potential contaminants from the copurified RBPs, requirement of isotope manipulation, and potential loss of information during a tedious experimental procedure. Here we describe a modified CLIP-seq method called FbioCLIP-seq using the FLAG-biotin tag tandem purification. Through tandem purification and stringent wash conditions, almost all the interacting RNA-binding proteins are removed. Thus, the RNAs interacting indirectly mediated by these copurified RBPs are also reduced. Our FbioCLIP-seq method allows efficient detection of direct protein-bound RNAs without SDS-PAGE and membrane transfer procedures in an isotope-free and protein-specific antibody-free manner.

Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

RNA and RNA-binding proteins (RBPs) control multiple biological processes. The spatial and temporal arrangement of RNAs and RBPs underlies the delicate ...