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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Research

JoVE Journal

Genetics

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular gene&#39;s function in the living organism. In this protocol the OSCAR method of precise and rapid deletion plasmid construction is described. OSCAR relies on the cloning system in which a single recombinase reaction is carried out containing the purified PCR-amplified 5&#39; and 3&#39; flanks of the gene of interest and two plasmids, pA-Hyg OSCAR (the marker vector) and pOSCAR (the assembly vector). Confirmation of the correctly assembled deletion vector is carried out by restriction digestion mapping followed by sequencing. Agrobacterium tumefaciens is then used to mediate introduction of the deletion construct into fungal spores (referred to as ATMT). Finally, a PCR assay is described to determine if the deletion construct integrated by homologous or non-homologous recombination, indicating gene deletion or ectopic integration, respectively. This approach has been successfully used for deletion of numerous genes in Verticillium dahliae and in Fusarium verticillioides among other species.

Rapid Deletion Production in Fungi via Agrobacterium Mediated Transformation of OSCAR Deletion Constructs

Gene deletion mutants generated through homologous recombination are the gold standard for gene function studies. The OSCAR (One Step Construction of Agrobacterium-Recombination-ready-plasmids) method for rapid generation of deletion constructs is described. Agrobacterium mediated fungal transformation follows. Finally, a PCR based confirmation method of gene deletions in fungal transformants is presented.

Precise deletion of gene(s) of interest, while leaving the rest of the genome unchanged, provides the ideal product to determine that particular ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Targeted genome editing in the model system Danio rerio (zebrafish) has been greatly facilitated by the emergence of CRISPR-based approaches. Herein, we describe a streamlined, robust protocol for generation and identification of CRISPR-derived nonsense alleles that incorporates the heteroduplex mobility assay and identification of mutations using next-generation sequencing.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

A Fast Silver Staining Protocol Enabling Simple and Efficient Detection of SSR Markers using a Non-denaturing Polyacrylamide Gel

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver staining is a widely used method for the detection of SSR markers in a polyacrylamide gel. However, conventional protocols for silver staining are technically demanding and time-consuming. Like many other biological laboratory techniques, silver staining protocols have been steadily optimized to improve detection efficiency. Here, we report a simplified silver staining method that significantly reduces reagent costs and enhances the detection resolution and picture clarity. The new method requires two major steps (impregnation and development) and three reagents (silver nitrate, sodium hydroxide, and formaldehyde), and only 7 min of processing for a non-denaturing polyacrylamide gel. Compared to previously reported protocols, this new method is easier, quicker and uses fewer chemical reagents for SSR detection. Therefore, this simple, low-cost, and effective silver staining protocol will benefit genetic mapping and marker-assisted breeding by a quick generation of SSR marker data.

Here, we report a simple and low-cost silver staining protocol which requires only three reagents and 7 min of processing, and is suitable for fast generation of high-quality SSR data in the genetic analysis.

Simple Sequence Repeat (SSR) is one of the most effective markers used in plant and animal genetic research and molecular breeding programs. Silver ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing in C. albicans requires Cas9, guide RNA, and repair template. A plasmid expressing a yeast codon optimized Cas9 (CaCas9) has been generated. Guide sequences directly upstream from a PAM site (NGG) are cloned into the Cas9 expression vector. A repair template is then made by primer extension in vitro. Cotransformation of the repair template and vector into C. albicans leads to genome editing. Depending on the repair template used, the investigator can introduce nucleotide changes, insertions, or deletions. As C. albicans is a diploid, mutations are made in both alleles of a gene, provided that the A and B alleles do not harbor SNPs that interfere with guide targeting or repair template incorporation. Multimember gene families can be edited in parallel if suitable conserved sequences exist in all family members. The C. albicans CRISPR system described is flanked by FRT sites and encodes flippase. Upon induction of flippase, the antibiotic marker (CaCas9) and guide RNA are removed from the genome. This allows the investigator to perform subsequent edits to the genome. C. albicans CRISPR is a powerful fungal genetic engineering tool, and minor alterations to the described protocols permit the modification of other fungal species including C. glabrata, N. castellii, and S. cerevisiae.

CRISPR-mediated Genome Editing of the Human Fungal Pathogen Candida albicans

Efficient genome engineering of Candida albicans is critical to understanding the pathogenesis and development of therapeutics. Here, we described a protocol to quickly and accurately edit the C. albicans genome using CRISPR. The protocol allows investigators to introduce a wide variety of genetic modifications including point mutations, insertions, and deletions.

This method describes the efficient CRISPR mediated genome editing of the diploid human fungal pathogen Candida albicans. CRISPR-mediated genome editing ...