Name: lentiviral mediated production of transgenic mice: a simple and highly efficient method for direct study of founders
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We thank Magali Dumont and Rolando Meloni for critical reading of the manuscript and the iVector and Phenoparc ICM Cores for technical assistance in lentiviral vector production and animal housing respectively. This work was supported by the Institut Hospitalo-Universitaire de Neurosciences Translationnelles de Paris, IHU-A-ICM, Investissements d&#39;Avenir ANR-10-IAIHU-06. P.R. received funding for the Association de Langue Fran&#231;aise pour l&#39;Etude du Diab&#232;te et des Maladies M&#233;taboliques (ALFEDIAM) and a joint JDRF / INSERM grant.

All procedures that include animal work have obtained ethical approval and have been authorized by the French Ministry of Research and Education under number APAFIS#5094-20 16032916219274 v6 and 05311.02. The ICM animal facility PHENOPARC has been accredited by the French Ministry of Agriculture under the accreditation number B75 13 19. The overall protocol requires performing each procedure within a precise time frame that is summarized in in Figure 1.
1. Animal Pu...

<ol>
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The perivitelline injection of lentiviral vectors in fertilized oocytes described here resulted in the production of transgenic embryos that yielded more than 70% of transgenic embryos relative to total number of collected embryos. This result is consistent with previous reports and exemplifies the specificity of the procedure2,7,10,11,12. When comparing all t...

The pioneering work of Gordon et al. in 1980 showed that after implantation in pseudopregnant mice, the plasmid DNA injection into the male pronuclei of fertilized oocytes can yield the production of transgenic animals that integrated the plasmid DNA1. The demonstration that transgenic mammals can be generated had an enormous impact on global life sciences, opening the way to novel fields of research both for basic sciences and translational biomedical sciences. In the past four decades, DNA microinjection has become a routine practice. Although an enormous number of transgenic mice have been produced, the standard method is not fully usable for all mouse strains and requires time consuming backcrosses2,3. Its application to other species remains challenging4 and the overall transgene integration yield is limited to a few percentage of born animals5. In addition, the efficacy of transgene integration represents the limiting factor that explains the poor overall yield of pronuclear DNA injection. In this respect, integrative viral vectors are the most efficient tools to cargo and integrate transgenes and thus could provide new means to significantly increase integration yield, the only limitation being that the transgene size that cannot exceed 10 kb6.
Lentiviral vectors pseudo-typed with the envelop protein of the Vesicular Stomatitis Virus (VSV) are pantropic and highly integrative gene transfer tools and can be used to transduce fertilized oocytes7. The zona pellucida surrounding the oocytes is a natural virus barrier and needs to be passed to allow transduction with the lentiviral vectors. Transgenic animals have been generated by transducing fertilized oocytes after micro-drilling or removing of the zona pellucida8,9. However, injection beneath the zona pellucida in the perivitelline space appears to be the simplest method to transduce the fertilized eggs as initially described by Lois and colleagues7.
The perivitelline injection of lentiviral vectors allows high yields in the production of transgenic animals that are above 70% of born animals. Such yield is over 10-fold higher than the best yield that can be achieved using standard pronuclei DNA injection7,10,11. In this context, a single session of injections will generate at least 50 transgenic founders (F0). The large number of founders is, therefore, compatible with phenotyping of the transgene effect directly performed on F0 mice without the need to generate transgenic mouse lines. This advantage allows for rapid screening of the transgene effect and is specifically adapted to perform in vivo gain and loss of function studies within weeks. In addition, regulatory DNA elements can also be rapidly screened to map enhancers and DNA motifs bound by transcription factors11,12. With pronuclear injections, transgenes usually integrate as multiple copies in a unique locus. With lentiviral vectors, integration occurs in multiple loci as a single copy per locus10,13. Therefore, the multiplicity of integrated loci is most likely associated to the very high expression penetrance observed in the transgenic founders, which makes the new generated model more robust.
Importantly, when using pronuclear injection of DNA, visualization of pronuclei during the procedure is absolutely required. This technical limitation prevents the usage of fertilized oocytes originating from a large variety of mouse strains. Therefore, production of a transgenic model in a specific strain for which pronuclei are invisible requires the production of animals in a permissive strain followed by at least 10 successive backcrosses to transfer the transgene in the desired mouse strain. With the lentiviral vector injections, perivitelline space is always visible and the injection does not require highly specific skills. As an example, NOD/SCID transgenic mice that are not appropriate for pronuclei injection have been obtained with the viral vector injections14.
Here, a comprehensive protocol is presented to allow simple production of transgenic mice using lentiviral vector injections in the perivitelline space of a one cell stage embryo. Transgene expression controlled with either ubiquitous or cell specific promoters is described in detail.
The pTrip &#916;U3 lentiviral backbone was used in this study15. This vector allows for producing replication defective lentiviral vectors in which the U3 sequence has been partially deleted to remove U3 promoter activity and generate a self-inactivating vector (SIN)16. Lentiviral vector stocks were produced by transient transfection of HEK-293T cells with the p8.91 encapsulation plasmid (&#916;Vpr &#916;Vif &#916;Vpu &#916;Nef)6, the pHCMV-G encoding the vesicular stomatitis virus (VSV) glycoprotein-G17, and the pTRIP &#916;U3 recombinant vector. The detailed production procedure is provided as supplemental methods.
Production of high titer lentiviral vector stocks is performed under Biosafety Level II conditions (BSL-2). This is true for most transgenes except for oncogenes that have to be produced in BSL-3. Therefore, production in BSL-2 conditions for most cases is sufficient. In addition, the use and the production are usually disconnected for most national regulatory agencies dealing with genetically modified organisms (GMO). Limited amounts of replication incompetent SIN lentiviral vectors (below 2 &#181;g of p24 capsid protein) can be used under BSL-1 conditions as described by the French GMO agency in agreement with the European Union recommendations.

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">PMSG 50UI</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">G4527</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">hCG 5000UI</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">CG5-1VL</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">NaCl</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">7982</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">100 mm petri dish</td>
			<td style="width:143px;">Dutsher</td>
			<td style="width:107px;">353003</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">4 wells Nunc dish</td>
			<td style="width:143px;">Dutsher</td>
			<td style="width:107px;">56469</td>
			<td>IVF dish</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">M2 medium</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">M7167</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">M16 medium</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">M7292</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">0,22 &#181;m Syringe filter</td>
			<td style="width:143px;">Dutsher</td>
			<td style="width:107px;">146611</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Hyaluronidase Enzyme 30mg</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">H4272</td>
			<td>mouse embryo tested</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin serynge</td>
			<td>VWR</td>
			<td>613-3867</td>
			<td>Terumo Myjector</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Curved forceps</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">2183</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Curved scissors</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">MC26</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:229px;">Aspirator tube assemblies for calibrated microcapillary pipettes</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">A5177-5EA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Borosilicate glass capillaries</td>
			<td style="width:143px;">Harvard apparatus</td>
			<td style="width:107px;">GC 100-10</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Horizontal micropipette puller</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:107px;">PN-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Microforge</td>
			<td style="width:143px;">Narishige</td>
			<td style="width:107px;">MF-900</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:229px;">Inverted microscope</td>
			<td style="width:143px;">Nikon</td>
			<td style="width:107px;">Transferman NK2 5188</td>
			<td style="width:164px;">Hoffman modulation contrast illumination is required</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Micromanipulator</td>
			<td style="width:143px;">Eppendorf</td>
			<td style="width:107px;">Celltram air</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Controler of holding pipet</td>
			<td style="width:143px;">Eppendorf</td>
			<td style="width:107px;">Femtojet</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Mineral oil</td>
			<td style="width:143px;">Sigma</td>
			<td style="width:107px;">M8410</td>
			<td>mouse embryo tested</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:229px;">Microinjector</td>
			<td style="width:143px;">Eppendorf</td>
			<td style="width:107px;">Femtojet</td>
			<td style="width:164px;">Can be used to inject DNA or viral vectors</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Dumont # 5 forceps</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">MC 40</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">vannas micro scissors</td>
			<td style="width:143px;">Moria</td>
			<td style="width:107px;">9600</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isoflurane</td>
			<td>centravet</td>
			<td>ISO005</td>
			<td>ISO-VET 100% 250ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">ocrygel</td>
			<td style="width:143px;">centravet</td>
			<td style="width:107px;">OCR002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:229px;">Povidone iodure</td>
			<td style="width:143px;">centravet</td>
			<td style="width:107px;">VET001</td>
			<td>vetedine 120ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Buprenorphine</td>
			<td>centravet</td>
			<td>BUP002</td>
			<td>Buprecare 0,3Mg/ml 10ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tris-HCl</td>
			<td>Sigma</td>
			<td>T5941</td>
			<td>Trizma hydrochloride</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA</td>
			<td>Sigma</td>
			<td>E9884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SDS</td>
			<td>Sigma</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td>powder</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">proteinase K</td>
			<td>Sigma</td>
			<td>P2308&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">oneTaq kit</td>
			<td>NEB</td>
			<td>M0480L</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Primers</td>
			<td>Eurogentec</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Strip of 8 PCR tube</td>
			<td>4titude</td>
			<td>4ti-0781</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well thermal cycler</td>
			<td>Applied Biosystems</td>
			<td>4375786</td>
			<td>Veriti</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Genomic DNA mini kit</td>
			<td>invitrogen</td>
			<td>K1820-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nanodrop 2000</td>
			<td>Thermo Scientific</td>
			<td>ND-2000C&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR Master mix</td>
			<td>Roche</td>
			<td>4887352001</td>
			<td>SYBR Green&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell plate 384</td>
			<td>Roche</td>
			<td>5217555001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">qPCR instrument 384 well</td>
			<td>Roche</td>
			<td>5015243001</td>
			<td>LightCycler 480</td>
		</tr>
	</tbody>
</table>

lentiviral mediated production of transgenic mice: a simple and highly efficient method for direct study of founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Transgenic animals were generated using the protocol presented here. Representative results both ubiquitous and cell type specific transgene expression are illustrated.
Constitutive expression of transgenes
Ubiquitous promoters are basic research tools to express transgenes in a sustained and efficient manner. Such promoters are used for a very large v...

Watch this Scientific Journal Video about Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders at JoVE.com

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

This method allows rapid in vivo screening in mice, of the effect of a transgene, and is specifically adapted to perform gain or loss of function studies, as well an answer and DNA motive mapping. The main advantage of this technique, is that the prediction use of transgenic animals are above 70%of newborn and can be applied to any mouse strains including transgenic animals. After euthanizing the mice, collect the oviducts.To begin the surgery, use scissors to make a large horizontal incision into the abdominal cavity. Then, locate the oviduct between the uterus and the ovary. Next, use curved forceps to remove the mesometrium and the membrane with the prominent blood vessels.Then, separate the oviduct from the ovary and dissect the oviduct from the ovary. Then, tug on the oviduct and cut away from the uterus. Transfer all the collected oviducts to M2 medium at room temperature.When handling the oviducts, be careful to avoid touching the swollen ampulla loaded with fertilized eggs. Next, add a 100 microliter drop of hyaluronidase working solution to a 10 centimeter dish. Then transfer the two oviducts into it, to remove the cumulus cells from the eggs.Under a stereomicroscope, use a pair of insulin syringes to secure the oviduct while tearing up the ampulla and dispersing the fertilized eggs. Then, use a prepared glass pipette with tubing and a mouthpiece to aspirate all the eggs. Using this tool, transfer them between six drops of M2 medium to wash off the surrounding cumulus cells.Then, transfer the washed eggs into a 10 millimeter well, in a four well plate, filled with preheated with M16. Then, transfer the dish to a humidified 37 degree Celsius incubator. In preparation, centrifuge the prepared lentiviral vector suspension at 160 g for two minutes, to pellet the debris often present in lentiviral stock.In a class two safety cabinet, transfer the supernatants to a new 0.5 milliliter tube. Next, load one microliter of supernatant, into an injection pipette. Then, attach the injection pipette to the right side micromanipulater.And attach the holding pipette to the left side micromanipulator. Now, dispense eight microliters of M2 medium in the center of the depression slide. And cover with light parafin oil to avoid evaporation.Then, place 20 eggs into the shallow locations in the drop without creating bubbles. Next, make sure that the tip of the injection pipette is open. If not, tap the injection pipette with the holding pipette to open it.Then, set the microinjector for 20 second injections. Now, using the holding pipette and manual pressure aspirate a fertilized egg that contains two pronuclei and two polar bodies. Then, using the injection pipette inject the perivitelline space of the egg.Do not touch the plasma membrane. Adjust the pressure so that the perivitelline space is filled with solution. The pressure should not exceed 600 hecto pascals.Immediately after injecting the 20 eggs, transfer the batch into a bath of preheated M16 medium. And incubate them for at least 30 minutes before implanting them. 16 hours after mating normal females to males with vasectomies, check for copulation plugs.Use these females for the implantation. Next, make implantation pipettes with internal diameter of about 150 microns. And with flame polished tips that are about four to five millimeters long.Load a pipette with embryo tested, light parafin oil to just above the shoulder point. Then, aspirate a small air bubble and further load the pipette with M2 medium. Next, aspirate a second air bubble.Now, draw up the embryos into the pipette one behind the other, minimizing the volume of medium between the embryos. Finish, by aspirating a very small drop of parafin oil just one embryo wide. Preparation of the reimplantation pipette is key and needs a lot of practice.Eggs in the pipette must be aligned with practically no medium in between them. Next, after anesthetizing the mouse and confirming the anesthesia with a toe pinch Shave off two, two centimeter strips of hair along the spinal cord at the level of the last rib. Now, place the mouse on a heating pad on a sterile field and drape it, leaving a window to the exposed skin.Next, sanitize the exposed skin with alternating scrubs of iodoprovidone and 70 percent ethanol. For the implantation, first make a one centimeter transverse incision, and then slide the skin laterally until the orange colored ovary is visible through the body wall. Next, make a five millimeter incision with the fine scissors and pick up the fat pad with an atraumatic bulldog clamp.Once the pad is gripped, pull out the ovary, the oviduct and the top of the uterus. Next, locate the ampulla and make a hemisection with vannas scissor, on the oviduct segment that links the ovary to the ampulla. Then, introduce the transfer embryo pipette and eject the eggs.Stopping at the first air bubble after the eggs have been released. Have the required materials prepared to repeat the procedure on the second oviduct. And once completed, close the skin up with wound clips.Immediately after closing the skin, intraperitonealy inject analgesic and place the animal in the recovery chamber with a 39 degree Celsius heating element. Monitor until it is fully awake. Transgenic animal were generated using the presented method.A high titer lentiviral vectors were produced using a 2.3 kilo base enhancer for Neurogenin-3 to drive eGFP. Or as a control, a fragment of chromosome six to drive eGFP. Integration into embryos was fairly successful.From the embryos, the pancreatic bud was sectioned and immunostained to look for localized expression of eGFP. Over 90 percent of the vectors, with the Neurogenin-3 enhancer, expressed eGFP in Neurog3 expressing cells. However, none of the integrated control vectors expressed eGFP in the pancreatic bud.Transgene integration sites were evaluated by quantitative PCR, using the CDX2 gene as a control. The average number of integration sites was about 20 using the Neurogenin-3 construct, or 10 using the control construct. Curiously, the viral transduction titer used for the Neurogenin-3 construct, was about double of the control vector.Suggesting a relationship between the transduction titer and the number of integrations. After watching this video, you should have a good understanding of how to generate large numbers of founder transgenic embryos using perivitelline injection of lentiviral vectors with high efficacy. Once mastered, this technique can produce up to 50 founder animals within a single day of microinjection, if it's performed properly.Following this procedure, other methods like titer pronuclear DNA injection can be performed, in order to establish transgenic mouse lines. Don't forget that working with lentiviral vectors is subjected to authorization from your local GMO committee. These vectors can be extremely hazardous and precaution should always be taken while performing this procedure.

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