This work was supported by National Science Fund for Distinguished Young Scholars (81625002), National Natural Science Foundation of China (81470389, 81270282, 81601238), Program of Shanghai Academic Research Leader (18XD1402400), Shanghai Municipal Education Commission Gaofeng Clinical Medicine Grant Support (20152209), Shanghai Shenkang Hospital Development Center (16CR3034A), Shanghai Jiao Tong University (YG2013MS42), Shanghai Jiao Tong University School of Medicine (15ZH1003 and 14XJ10019), Shanghai Sailing Program (18YF1413000), and Postgraduate Innovation Program of Bengbu Medical College (Byycx1722). We thank Dr. Erhe Gao for his previous help in our lab.

All animal experiments were carried out according to the National Institutes of Health Guidelines on the Use of Laboratory Animals, and were approved by the Institute&#39;s Animal Ethics Committee. Male C57BL/6J mice (aged 8 - 10 weeks) were used for all the experiments. Mice were housed under pathogen-free conditions at 24 &#176;C &#177; 4 &#176;C, under a 12-h light/dark cycle, with free access to water and food.
1. Preparation of Adenovirus Solution
<ol>
	<li>Upon arrival of the purified adenovirus solution, store the solution in a -80 &#176;C freezer. 
	NOTE: The adenovirus injection was performed in viable mice. To ensure the sterility of the adenovirus vector, the adenovirus (encoding luciferase, VDR, or shRNA targeting VDR) were prepared and purified commercially9,10.</li>
	<li>On the day of operation, take out the purified adenovirus solution (3 x&#160;1010 plaque forming units [pfu]/mL) from the -80 &#176;C freezer, and thaw adenovirus vector solution on ice.</li>
	<li>Put on a facemask, sterile gloves, and sterile gown.</li>
	<li>Prepare a 50-&#181;L Hamilton syringe which has been sterilized the day before the operation. 
	NOTE: For sterilization, the Hamilton syringe was wrapped in gauze and placed in a high temperature/high pressure sterilizer. The mode of &#34;Sterilization for the solid&#34; was selected and sterilization was commenced by pressing the &#34;start&#34; button.</li>
	<li>After complete thawing of the purified adenovirus solution, spray the tube containing adenovirus solution with 75% ethanol, and move the adenovirus solution into a laminar flow sterile hood.</li>
	<li>In a laminar flow sterile hood, aspirate 50 &#181;L adenovirus solution using the Hamilton syringe without a needle, followed by attachment of a 30 G needle to the Hamilton syringe.</li>
	<li>Hold the syringe with the 30 G needle upward, and gently push the plunger of the syringe until the needle is filled with adenovirus solution (confirmed by the appearance of the first solution drops from the needle tip). 
	NOTE: It takes approximately 45 &#181;L solution to fill the 30 G needle, so now almost no solution is visible in the syringe.</li>
	<li>Use the above prepared syringe to carefully aspirate another 30 &#181;L adenovirus solution, and place the loosely capped syringe on ice for subsequent use.</li>
</ol>
2. Anesthesia and Operative Preparation
<ol>
	<li>Add 20 mL isoflurane into an isoflurane vaporizer, and connect the isoflurane vaporizer to an oxygen tank.</li>
	<li>Open the valve and let the oxygen flow from the oxygen tank to the isoflurane vaporizer. Maintain the oxygen flow rate at 2 L/min via an oxygen flow monitor in the isoflurane vaporizer.</li>
	<li>Induce anesthesia of the mouse with 4% isoflurane in 100% oxygen (2 L/min) for 2 min in a plastic cage connected to the isoflurane vaporizer.</li>
	<li>Take out the anesthetized mouse from the plastic cage, and secure it on a plastic platform in the prone position in a laminar flow sterile hood, and maintain anesthesia with 2% isoflurane in 100% oxygen (2 L/min) via a nose cone.</li>
	<li>Confirm adequate anesthesia by the absence of toe pinch response.</li>
	<li>Apply sterile ophthalmic cream to each eye to protect the corneas from drying.</li>
	<li>Shave the chest and the upper abdomen. Apply commercially available depilatory cream to the shaved site for 1 min. Remove the depilatory cream and remaining fur with wet gauzes.</li>
	<li>Sterilize the surgical site (left lower part of the chest) with 3 scrubs of iodine-chlorhexidine based antiseptic (e.g. entoiodine). Cover the surgical site with a sterile drape.</li>
</ol>
3. Intramyocardial Injection of Adenovirus in Mouse Heart
<ol>
	<li>Take off the gloves and put on a new pair of sterile gloves.</li>
	<li>Sterilize forceps, a micro-mosquito hemostat, a pair of surgical scissors, and a needle holder on the day before operation in a high temperature/high pressure sterilizer (see step 1.4 note).</li>
	<li>Make a 0.5-cm skin incision along the line connecting the xiphoid and axilla. Bluntly dissect the pectoral major and pectoral minor muscles with forceps and a micro-mosquito hemostat.</li>
	<li>Expose the intercostal spaces by retracting the pectoral major and pectoral minor muscles. Pierce through and open the fourth intercostal space (or the widest intercostal space by observation) with a micro-mosquito hemostat.</li>
	<li>Push the heart toward the incision to externalize the heart with the index finger of the non-dominant hand by gently pressing against the right side of the chest wall.</li>
	<li>Gently secure the externalized heart with the index finger and thumb of the non-dominant hand.</li>
	<li>Inject a total of 30 &#181;L adenovirus solution into the myocardium of the left ventricle in three sites (ventral, dorsal, and lateral wall of the left ventricle) via the Hamilton syringe filled with adenovirus (Figure 1C) with the dominant hand.</li>
	<li>After completion of injection, immediately place the heart back into the intrathoracic space.</li>
	<li>Manually evacuate the air in the intrathoracic space by gently pressing the chest wall toward the skin incision site. 
	Note: Successful air evacuation can be evidenced by flapping of the pectoral major and pectoral minor muscles caused by the expelled air.</li>
	<li>Close the skin by horizontal mattress suture with a 5-0 silk suture. 
	Note: The pectoral major and pectoral minor muscles should not be sutured, because they are only bluntly dissected and their anatomical structures are intact throughout the operation.</li>
</ol>
4. Postoperative Management
<ol>
	<li>Administer buprenorphine (3 mg/kg) twice daily via subcutaneous injection to reduce post-operative pain for the first 48 h after operation11.</li>
	<li>After operation, immediately maintain the mice on a heat pad (37 &#176;C) until fully recovered. Place the mouse back to the cage after it fully recovers.</li>
	<li>Sterilize the used Hamilton syringe and 30 G syringe needle in a high temperature/high pressure sterilizer (see step 1.4 note). Collect the sterilized 30 G syringe needle in a sharps container.</li>
</ol>
5. In Vivo Imaging for Measuring Cardiac Luciferase Expression
<ol>
	<li>On day 5 after adenovirus injection, prepare a fresh stock solution of luciferin at 15 mg/mL in Dulbecco&#39;s phosphate buffered saline (DPBS). Filter the solution through a 0.2 &#181;m filter.</li>
	<li>Intra-peritoneally inject the awake mice with the luciferin solution at 150 mg/kg body weight.</li>
	<li>Turn on the imaging system.</li>
	<li>After automatic self-test of the imaging system, select the imaging mode as &#34;Luminescent,&#34;&#160;and make the following settings: Exposure: Auto; Binning: 8; FStop: 1; Excitation: Block; Emission: Open.</li>
	<li>At 10 min after luciferin injection, anesthetize the mice by chloral hydrate injection (300 mg/kg body weight). Confirm adequate anesthesia by the absence of toe pinch response.</li>
	<li>Secure the anesthetized mice on a heated imaging stage in the prone position, with the chest directed towards the camera.</li>
	<li>Collect images (1 image for each mouse) and quantify the intensity of signals by drawing identical circular measurement regions of interest (ROI) around the chest.</li>
	<li>After the collection of images, sacrifice the mice and collect the tissues as mentioned in the following section.</li>
</ol>
6. Harvesting Tissues
<ol>
	<li>At different time points after virus injection (Figure 1B), anesthetize the mice with 4% isoflurane, and maintain anesthesia with 2% isoflurane via a nose cone, and secure it on a plastic platform in the prone position.</li>
	<li>Make a midline ventral incision in the anterior neck. Expose the right common carotid artery by retracting the omohyoid and sternocleidomastoid muscles.</li>
	<li>Sacrifice the mice by transecting the right common carotid artery and draining the blood.</li>
	<li>Make a midline ventral incision in the abdomen. Cut the ribs from both sides of the rib cage along the midclavicular line, and transect the diaphragm.</li>
	<li>Expose the heart by lifting the xiphoid. Find the ascending aorta and gently remove the perivascular fat tissue.</li>
	<li>Cannulate the aorta and retrogradely perfuse the heart with a 30 G needle attached to a 1 mL syringe filled with 0.5 mL of 4 &#176;C phosphate buffer solution (PBS).</li>
	<li>After perfusion, excise the heart, and divide it into the left and right ventricles. Excise the liver, lung, and spleen.</li>
	<li>After two washes in cold PBS (4 &#176;C), store all the collected tissues separately in cryogenic vials in liquid nitrogen.</li>
</ol>
7. Determination of Protein Expression
<ol>
	<li>Prepare homogenization buffer by adding protease inhibitor cocktail into commercially available lysis buffer at a ratio of 1:100. Calculate the total volume based on the number of samples to be processed. Keep the prepared homogenization buffer at 4 &#176;C for subsequent use.</li>
	<li>Take out the tissues from the liquid nitrogen tank. Weigh the tissue on high-precision scales (approximately 60 mg for each piece of tissue).</li>
	<li>Transfer the tissue to a new 1.5-mL microcentrifuge tube. Immediately add 200 &#181;L homogenization buffer and precooled 1.5-mm steel balls (4 &#176;C) into the microcentrifuge tube.</li>
	<li>Secure the microcentrifuge tubes in precooled (4 &#176;C) metal holders in an automatic tissue grinding machine, and commence homogenization by starting the machine with the following settings: Frequency: 70 Hz; time: 120 s.</li>
	<li>After homogenization, centrifuge the homogenate (16,000 &#215; g, 4 &#176;C), and carefully transfer the supernatant to a new 1.5-mL tube with a 100 &#181;L pipette.</li>
	<li>Add loading buffer solution (5x)&#160;to the protein supernatant at a ratio of 1:4, and denature the protein at 100 &#176;C for 5 min.</li>
	<li>The protein expression levels in the heart and other organ tissues are further monitored by Western blot analysis (representative results are shown in Figure 2C and Figure 3) according to the protocol previously described12.</li>
</ol>

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The authors have nothing to disclose.

The current report demonstrates a modified technique for intramyocardial injection of viral vectors for cardiac gene manipulation, which was modified from a method for myocardial infarction induction by Gao et al.13 Currently, in vivo characterization of specific gene functions most often involve the generation of knockout or transgenic mice3,14,15,16<s...

Cardiac disease is the leading cause of morbidity and mortality worldwide1,2. The lack of effective therapeutic strategies for life-threatening heart conditions including myocardial infarction and heart failure attracts intensive exploration of underlying pathomechanisms and identification of novel therapeutic options3. For these scientific explorations, cardiac-specific gene manipulation is widely used4,5. Cardiac gene manipulation can be achieved by genome editing using the powerful transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) tools, or by delivery of ectopic genetic materials (e.g., virus vectors carrying genes encoding proteins of interest)6. Though genome editing allows precise and spatiotemporal genome modifications in living mice, it is still a time-consuming and labor-intensive practice6. Alternatively, cardiac-specific gene manipulation by virus vector or small interfering RNA (siRNA) complex delivery are routinely performed6.
Virus vector delivery to the adult mouse heart is achieved by roughly two strategies: systemic or local injection. Systemic injection of cardiotropic serotype of AAVs such as AAV9 is noninvasive for cardiac specific gene manipulation7. However, this method requires a relatively high amount of vector necessary for efficient transduction and gene expression, and may result in significant transduction of nontarget organs such as the muscle and liver7. Local virus injection is achieved by intramyocardial injection or intracoronary delivery7. Intracoronary delivery leads to a more even distribution of virus within the heart compared to intramyocardial injection. However, the disadvantages of this technique are the rapid wash out of viral vectors to the systemic circulation and transduction in nontarget organs8, and its requirement of devices for pressure measurement during the operation. By contrast, intramyocardial injection enables better virus retention in the myocardium as well as site specific delivery, but it fails to evenly distribute viral vector7. For small animals, intracoronary delivery is technically difficult to perform, while systemic AAV9 injection and intramyocardial injection are more commonly practiced4,5,7. Though systemic injection is easy to perform, conventional intramyocardial injection requires mechanical ventilation and thoracotomy, causes extensive tissue damage, and is time-consuming.
In this report, we described an easy, time-saving, and highly efficient method for intramyocardial injection. Adenovirus encoding luciferase and VDR, or shRNA targeting VDR, was injected to manipulate cardiac gene expression. Once mastered, this method can be used for gene manipulation, as well as injection of cells or other materials in the mouse heart.

<table border="0" cellpadding="0" cellspacing="0" width="914">
	<tbody>
		<tr height="40">
			<td colspan="4" height="40" style="height:40px;width:916px;">Equipments</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Laminar flow sterile hood</td>
			<td style="width:267px;">Fengshi Animal Experimental&#160; Equipment Techonology Co., Ltd. (Soochow, China)</td>
			<td style="width:143px;">FS-CJ-2F</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Centrifuge</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">75005282</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Tissue grinding machine</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;">Scientz-48</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">High temperature/high pressure sterilizer</td>
			<td style="width:267px;">Hirayama (Saitama, Japan)</td>
			<td style="width:143px;">HVE-50</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Isoflurane vaporizer&#160;</td>
			<td style="width:267px;">Matrix (Orchard Park, USA)</td>
			<td style="width:143px;">VIP3000</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">IVIS&#160; Lumina III imaging system</td>
			<td style="width:267px;">PerkinElmer (Waltham, USA)</td>
			<td style="width:143px;">CLS136334</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Precision&#160;balance</td>
			<td style="width:267px;">Sartorius (G&#246;ttingen, Germany)</td>
			<td style="width:143px;">28091873</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Instruments&#160;</td>
			<td style="width:267px;"></td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (100 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000059</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Eppendorf pipette (10 &#181;L)</td>
			<td style="width:267px;">Eppendorf (Westbury, USA)</td>
			<td style="width:143px;">&#160;4920000113</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Forceps</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp.&#160;</td>
			<td style="width:143px;">JD4020</td>
			<td style="width:156px;">Curved tip</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Hamilton syringe</td>
			<td style="width:267px;">Hamilton (Nevada, USA)</td>
			<td style="width:143px;">80501</td>
			<td style="width:156px;">Volume 50 &#956;L</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Micro-mosquito hemostat</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">13011-12</td>
			<td style="width:156px;">Curved, tip width 1.3mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Needle holder&#160;</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;">J32110</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Surgical scissors</td>
			<td style="width:267px;">F.S.T (Foster City, USA)</td>
			<td style="width:143px;">14002-12</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">1-mL Syringe</td>
			<td style="width:267px;">WeiGao Group Medical Polymer Co.,Ltd. (ShangDong, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Materials and reagents</td>
			<td style="width:267px;"></td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-GAPDH antibody</td>
			<td style="width:267px;">CST (Danvers,&#160; USA)</td>
			<td style="width:143px;">#2118</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-Luciferase antibody</td>
			<td style="width:267px;">Abcam (Cambridge, UK)</td>
			<td style="width:143px;">ab187340</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-rabbit IgG</td>
			<td style="width:267px;">CST (Danvers,&#160; USA)</td>
			<td style="width:143px;">&#160;#7074</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Anti-VDR antibody</td>
			<td style="width:267px;">Abcam (Cambridge, UK)</td>
			<td style="width:143px;">&#160;ab109234</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Buprenorphine</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">PA175056</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Chloralic hydras</td>
			<td style="width:267px;">LingFeng Chemical (ShangHai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Cryogenic Vials</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">375418</td>
			<td style="width:156px;">1.8 mL&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Depilatory cream</td>
			<td style="width:267px;">Veet (Shanghai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Dulbecco&#39;s phosphate buffered saline&#160;</td>
			<td style="width:267px;">Gibco (Grand Island,&#160; USA)</td>
			<td style="width:143px;">14040133</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Entoiodine</td>
			<td style="width:267px;">LiKang (Shanghai, China)</td>
			<td style="width:143px;">310132</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">EP tube</td>
			<td style="width:267px;">Sarstedt (Newton, USA)</td>
			<td style="width:143px;">PCR001</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Filter</td>
			<td style="width:267px;">Millipore (Bedford, USA)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">Pore size 0.2 &#181;m&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Isoflurane</td>
			<td style="width:267px;">Yipin Pharmaceutical Company (Hebei, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Luciferin</td>
			<td style="width:267px;">Promega (Madison, USA)</td>
			<td style="width:143px;">P1041</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Lysis buffer for western&#160; blot</td>
			<td style="width:267px;">Beyotime (Shanghai, China)</td>
			<td style="width:143px;">P0013J</td>
			<td style="width:156px;">Without inhibitors</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Ophthalmic cream</td>
			<td style="width:267px;">Apex Laboratories ( Melbourne, Australia))</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">PBS</td>
			<td style="width:267px;">Gibco (Grand Island,&#160; USA)</td>
			<td style="width:143px;">10010023</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Protease inhibitor cocktail</td>
			<td style="width:267px;">Thermo Scientific (Waltham, USA)</td>
			<td style="width:143px;">78438</td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">5-0 silk suture</td>
			<td style="width:267px;">Shanghai Medical Instruments (Group) Ltd., Corp. (Shanghai, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Steel ball</td>
			<td style="width:267px;">Scientz Biotechnology Co., Ltd. (Ningbo, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">Width 1.5 mm</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Syringe needle</td>
			<td style="width:267px;">Kindly Medical Devices Co., Ltd. (Zhejiang, China)</td>
			<td style="width:143px;"></td>
			<td style="width:156px;">30 gauge&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:351px;">Warm mat</td>
			<td style="width:267px;">Warmtact Electrical Heating Technology Co., Ltd. (Guangdong, China )</td>
			<td style="width:143px;">NF-GNCW</td>
			<td style="width:156px;"></td>
		</tr>
	</tbody>
</table>

a simple and efficient method for in vivo cardiac-specific gene manipulation by intramyocardial injection in mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.

The experiment protocol and some of the key steps for the reported method are shown in Figure 1. At 5 days after intramyocardial injection of adenovirus encoding luciferase (Adv-luc), in vivo imaging in adv-luc injected mice indicated robust overexpression of luciferase specifically in the heart (Figure 2A, B), which was confirmed by Western blot analysis (Figure 2C), sugges...

Watch this Scientific Journal Video about A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice at JoVE.com

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. ...

a-simple-efficient-method-for-vivo-cardiac-specific-gene-manipulation

Research

JoVE Journal

Biology

15.7K Views.  The First Affiliated Hospital of Bengbu Medical College. Here we present a protocol for cardiac-specific gene manipulation in mice. Under anesthesia, the mouse hearts were externalized through the fourth intercostal space. Subsequently, adenoviruses encoding specific genes were injected with a syringe into the myocardium, followed by protein expression measurement via in vivo imaging and Western blot analysis.

Video: A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Intravenous Injections in Neonatal Mice

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are technically feasible and routine. However, some mouse models can have early onset of disease with a rapid progression that makes administration of potential therapies difficult. The temporal (or facial) vein is just anterior to the ear bud in mice and is clearly visible for the first two days after birth on either side of the head using a dissecting microscope. During this window, the temporal vein can be injected with volumes up to 50 &#956;l. The injection is safe and well tolerated by both the pups and the dams. A typical injection procedure is completed within 1-2 min, after which the pup is returned to the home cage. By the third postnatal day the vein is difficult to visualize and the injection procedure becomes technically unreliable. This technique has been used for delivery of adeno-associated virus (AAV) vectors, which in turn can provide almost body-wide, stable transgene expression for the life of the animal depending on the viral serotype chosen.

Animal models of pediatric disease can experience early onset and aggressive disease progression. Clinically relevant therapy delivery to young mouse models can be technically difficult. This protocol describes a non-invasive intravenous injection method for newborn mice within the first two postnatal days of life.

Intravenous injection is a clinically applicable manner to deliver therapeutics. For adult rodents and larger animals, intravenous injections are ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.