Name: mouse in vivo placental targeted crispr manipulation
Uploaded: 2023-04-14T14:40:00
Description: Watch this Scientific Journal Video about Mouse In Vivo Placental Targeted CRISPR Manipulation at JoVE.com

The authors acknowledge the following funding sources: R01 MH122435, NIH T32GM008629, and NIH T32GM145441. The authors thank Dr. Val Sheffield and Dr. Calvin Carter&#39;s labs at the University of Iowa for the use of their surgery room and equipment, as well Dr. Eric Van Otterloo, Dr. Nandakumar Narayanan, and Dr. Matthew Weber for their assistance with microscopy. The authors also thank Dr. Sara Maurer, Maya Evans, and Sreelekha Kundu for their assistance with the pilot surgeries.

All procedures were performed in accordance and compliance with federal regulations and University of Iowa policy and were approved by Institutional Animal Care and Use Committee.
1. Animals and husbandry
<ol>
	<li>Keep the animals in a 12 h daylight cycle with food and water ad libitum.</li>
	<li>Use CD-1 female mice aged 8-15 weeks. Use the presence of a copulatory plug to identify E0.5.</li>
	<li>On E0.5, singly house the pregnant dams.</li>
</ol>
<p class="jove_...

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+horizons+for+neurodevelopmental+disorders:+Placental+mechanisms.">Future horizons for neurodevelopmental disorders: Placental mechanisms.</a> Frontiers in Pediatrics. 9, 653230 (2021).</li><li>Woods, L., Perez-Garcia, V., Hemberger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+placental+development+and+its+impact+on+fetal+growth-new+insights+from+mouse+models.">Regulation of placental development and its impact on fetal growth-new insights from mouse models.</a> Frontiers in Endocrinology. 9, 570 (2018).</li><li>Goeden, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maternal+Inflammation+disrupts+fetal+neurodevelopment+via+increased+placental+output+of+serotonin+to+the+fetal+brain.">Maternal Inflammation disrupts fetal neurodevelopment via increased placental output of serotonin to the fetal brain.</a> Journal of Neuroscience. 36 (22), 6041-6049 (2016).</li><li>vander Bom, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+changing+epidemiology+of+congenital+heart+disease.">The changing epidemiology of congenital heart disease.</a> Nature Reviews Cardiology. 8 (1), 50-60 (2011).</li><li>Wilson, R. 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L., Leone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+Cre+recombinase+in+early+diploid+trophoblast+cells+of+the+mouse+placenta.">Expression of Cre recombinase in early diploid trophoblast cells of the mouse placenta.</a> Genesis. 45 (3), 129-134 (2007).</li><li>Zhou, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+expression+of+Cre+recombinase+provokes+placental-specific+DNA+recombination+in+transgenic+mice.">Targeted expression of Cre recombinase provokes placental-specific DNA recombination in transgenic mice.</a> PLoS One. 7 (2), e29236 (2012).</li><li>Wattez, J. S., Qiao, L., Lee, S., Natale, D. R. 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N., Crombleholme, T., Habli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral-mediated+placental+gene+transfer+of+IGF-1+corrects+placental+insufficiency+via+enhanced+placental+glucose+transport+mechanisms.">Adenoviral-mediated placental gene transfer of IGF-1 corrects placental insufficiency via enhanced placental glucose transport mechanisms.</a> PLoS One. 8 (9), e74632 (2013).</li><li>Jones, H., Crombleholme, T., Habli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+amino+acid+transporters+by+adenoviral-mediated+human+insulin-like+growth+factor-1+in+a+mouse+model+of+placental+insufficiency+in+vivo+and+the+human+trophoblast+line+BeWo+in+vitro.">Regulation of amino acid transporters by adenoviral-mediated human insulin-like growth factor-1 in a mouse model of placental insufficiency in vivo and the human trophoblast line BeWo in vitro.</a> Placenta. 35 (2), 132-138 (2014).</li><li>Song, A. 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K., Collen, D., VandenDriessche, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biosafety+of+adenoviral+vectors.">Biosafety of adenoviral vectors.</a> Current Gene Therapy. 3 (6), 527-543 (2003).</li><li>Evers, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+knockout+screening+outperforms+shRNA+and+CRISPRi+in+identifying+essential+genes.">CRISPR knockout screening outperforms shRNA and CRISPRi in identifying essential genes.</a> Nature Biotechnology. 34 (6), 631-633 (2016).</li><li>Rossant, J., Cross, J. 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B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+maternal+interleukin-17+and+autism-related+cortical+gene+expression,+neurobiology,+and+behavior.">Chronic maternal interleukin-17 and autism-related cortical gene expression, neurobiology, and behavior.</a> Neuropsychopharmacology. 45 (6), 1008-1017 (2020).</li><li>Liu, F., Huang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+gene+transfer+to+the+liver+following+systemic+administration+of+plasmid+DNA.">Electric gene transfer to the liver following systemic administration of plasmid DNA.</a> Gene Therapy. 9 (16), 1116-1119 (2002).</li><li>Kalli, C., Teoh, W. C., Leen, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+genes+via+sonoporation+and+electroporation.">Introduction of genes via sonoporation and electroporation.</a> Advances in Experimental Medicine and Biology. 818, 231-254 (2014).</li><li>Wu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+gene+editing+using+ribonucleoproteins+in+skin+stem+cells+of+recessive+dystrophic+epidermolysis+bullosa+mouse+model.">Efficient in vivo gene editing using ribonucleoproteins in skin stem cells of recessive dystrophic epidermolysis bullosa mouse model.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (7), 1660-1665 (2017).</li><li>Nakamura, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electroporation and Sonoporation in Developmental Biology. , (2009).</li><li>Bond, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+timing+and+coordination+of+neurogenesis+and+astrogenesis+in+developing+mouse+hippocampal+subregions.">Differential timing and coordination of neurogenesis and astrogenesis in developing mouse hippocampal subregions.</a> Brain Sciences. 10 (12), 909 (2020).</li><li>Kojima, Y., Tam, O. H., Tam, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timing+of+developmental+events+in+the+early+mouse+embryo.">Timing of developmental events in the early mouse embryo.</a> Seminars in Cell &amp; Developmental Biology. 34, 65-75 (2014).</li><li>Pennington, K. A., Schlitt, J. M., Schulz, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+primary+mouse+trophoblast+cells+and+trophoblast+invasion+assay.">Isolation of primary mouse trophoblast cells and trophoblast invasion assay.</a> Journal of Visualized Experiments. (59), e3202 (2012).</li><li>Mandegar, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+Interference+efficiently+induces+specific+and+reversible+gene+silencing+in+human+iPSCs.">CRISPR Interference efficiently induces specific and reversible gene silencing in human iPSCs.</a> Cell Stem Cell. 18 (4), 541-553 (2016).</li><li>Dai, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+CRISPRa+screen+identifies+putative+enhancers.">Inducible CRISPRa screen identifies putative enhancers.</a> Journal of Genetics and Genomics. 48 (10), 917-927 (2021).</li><li>Ursini, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placental+genomic+risk+scores+and+early+neurodevelopmental+outcomes.">Placental genomic risk scores and early neurodevelopmental outcomes.</a> Proceedings of the National Academy of Sciences of the United States of America. 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The placenta is a primary regulator of fetal growth, and as previously noted, changes in placental gene expression or function may significantly impact fetal development6. The protocol outlined here can be used to perform a targeted in vivo CRISPR manipulation of the mouse placenta using a relatively advanced surgical approach. This technique allows for a significant yield of viable embryos and their corresponding placentas that can be used for further study (Figure 6...

The placenta is an essential organ involved in the development of the fetus. The main role of the placenta is to provide essential factors and regulate the transfer of nutrients and waste to and from the fetus. Mammalian placentas are composed of both fetal and maternal tissue, which make up the fetal-maternal interface, and, thus, the genetics of both the mother and fetus impact function1. Genetic anomalies or impaired function of the placenta can drastically alter fetal development. Previous work has shown that placental genetics and development are associated with the altered development of specific organ systems in the fetus. Particularly, abnormalities in the placenta are linked with changes in the fetal brain, heart, and vascular system2,3,4,5.
The transport of hormones, growth factors, and other molecules from the placenta to the fetus plays a major role in fetal development6. It has been shown that altering the placental production of specific molecules can alter neurodevelopment. Maternal inflammation can increase the production of serotonin by altering tryptophan (TRP) metabolic gene expression in the placenta, which subsequently creates an accumulation of serotonin in the fetal brain7. Other studies have found placental abnormalities alongside heart defects. Abnormalities in the placenta are thought to contribute to congenital heart defects, the most common birth defect in humans8. A recent study has identified several genes that have similar cellular pathways in both the placenta and heart. If disrupted, these pathways could cause defects in both organs9. The defects in the placenta may exacerbate congenital heart defects. The role of placental genetics and function on specific fetal organ system development is an emerging field of study.
Mice have hemochorial placentas and other features of human placentas, which makes them highly useful models for studying human disease1. Despite the importance of the placenta, there is currently a lack of targeted in vivo genetic manipulations. Furthermore, there are currently more options available for knockouts or knockdowns than overexpression or gain-of-function manipulations in the placenta10. There are several transgenic Cre-expressing lines for placental-specific manipulation, each in different trophoblast lineages at different time points. These include Cyp19-Cre, Ada/Tpbpa-Cre, PDGFR&#945;-CreER, and Gcm1-Cre11,12,13,14. While these Cre transgenes are efficient, they may not be capable of manipulating some genes at specific time points. Another commonly used method to either knockout or overexpress placental gene expression is the insertion of lentiviral vectors into blastocyst culture, which causes a trophoblast-specific genetic manipulation15,16. This technique allows for a robust change in the placental gene expression early in development. The use of RNA interference in vivo has been sparsely utilized in the placenta. The insertion of shRNA plasmids can be performed similarly to the CRIPSR technique described in this paper. This has been done at E13.5 to successfully decrease PlGF expression in the placenta, with impacts on offspring brain vasculature17.
In addition to techniques that are primarily used for knockout or knockdown, inducing overexpression is commonly performed with adenoviruses or the insertion of an exogenous protein. The techniques used for overexpression have varying rates of success and have mostly been performed later in gestation. To investigate the role of insulin-like growth factor 1 (IGF-1) in placental function, an adenoviral-mediated placental gene transfer was performed to induce the overexpression of the IGF-1 gene18,19. This was performed late in mouse gestation on E18.5 via&#160;direct placental injection. To provide additional options and circumvent possible failures of established placental genetic manipulations, such as Cre-Lox combination failures, the possible toxicity of adenoviruses, and the off-target effects of shRNA, in vivo direct CRISPR manipulation of the placenta can be used20,21,22. This model was developed to address the lack of overexpression models and to create a model with flexibility.
This technique is based upon the work of Lecuyer et al., in which shRNA and CRISPR plasmids were targeted directly in vivo to mouse placentas to alter PlGF expression17. This technique can be used to directly alter placental gene expression using CRISPR manipulation at multiple time points; for this work, E12.5 was selected. The placenta has matured by this point and is large enough to manipulate, allowing for the insertion of a specific CRISPR plasmid on E12.5, which can have a significant impact on fetal development from mid to late pregnancy23,24. Unlike transgenic approaches, but similar to viral inductions or RNA interference, this technique allows for overexpression or knockout at particular time points using a relatively advanced surgical approach, thus avoiding possible impaired placentation or embryonic lethality from earlier changes. As only a few placentas receive the experimental or control plasmid within a litter, the approach allows for two types of internal controls. These controls are those injected and electroporated with the appropriate control plasmid and those that receive no direct manipulation. This technique was optimized to create an overexpression of the IGF-1 gene in the mouse placenta via a synergistic activation mediator (SAM) CRISPR plasmid. The IGF-1 gene was chosen, as IGF-1 is an essential growth hormone delivered to the fetus that is primarily produced in the placenta prior to birth25,26. This new placental-targeted CRISPR technique will allow for direct manipulation to help define the connection between placental function and fetal development.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >1.5 ml Tubes</td>
			<td >USA Scientific Inc</td>
			<td >1615-5500</td>
			<td ></td>
		</tr>
		<tr >
			<td >4% Paraformeldhyde (PFA) in PBS</td>
			<td >Thermo Fisher Scientific</td>
			<td >J61899.AP</td>
			<td></td>
		</tr>
		<tr >
			<td >96 Well plate</td>
			<td >Cornings</td>
			<td >3598</td>
			<td>For BCA kit</td>
		</tr>
		<tr >
			<td >Absorbent Underpads</td>
			<td >Fisher Scientific</td>
			<td >14-206-62</td>
			<td></td>
		</tr>
		<tr >
			<td >Activation Control Plasmid</td>
			<td >Santa Cruz Biotechnology</td>
			<td >sc-437275</td>
			<td>Dnase-free water provided for dilution</td>
		</tr>
		<tr >
			<td >AMV Reverse Transcriptase</td>
			<td >New England Biolabs</td>
			<td >M0277L</td>
			<td>Use for cDNA synthesis</td>
		</tr>
		<tr >
			<td >Anesthetic Gas Vaporizor</td>
			<td>Vetamac</td>
			<td >VAD-601TT</td>
			<td>VAD-compact vaporizer</td>
		</tr>
		<tr >
			<td >Artifical Tear Gel</td>
			<td >Akorn</td>
			<td >NDC 59399-162-35</td>
			<td></td>
		</tr>
		<tr >
			<td >BCA Protein Assay Kit</td>
			<td >Thermo Fisher Scientific</td>
			<td>23227</td>
			<td>Protein quantification</td>
		</tr>
		<tr >
			<td >Biovortexer</td>
			<td >Bellco Glass, Inc.</td>
			<td >198050000</td>
			<td>Hand-held tissue homogenizer</td>
		</tr>
		<tr >
			<td >CellSens Software</td>
			<td >Olympus</td>
			<td >V4.1.1</td>
			<td>Image processing to FISH images.</td>
		</tr>
		<tr >
			<td >Centrifuge 5810</td>
			<td >Eppendorf</td>
			<td >EP022628168</td>
			<td>Plate centrifuge</td>
		</tr>
		<tr >
			<td >Chloroform</td>
			<td >Thermo Fisher Scientific</td>
			<td >J67241-AP</td>
			<td>RNA isolation</td>
		</tr>
		<tr >
			<td >Cotton Tipped Applicators</td>
			<td >ProAdvantage</td>
			<td >77100</td>
			<td>Sterilize before use</td>
		</tr>
		<tr >
			<td >CRISPR/Cas9 Control Plasmid</td>
			<td >Santa Cruz Biotechnology</td>
			<td >sc-418922</td>
			<td>Dnase-free water provided for dilution</td>
		</tr>
		<tr >
			<td >CryoStat</td>
			<td >Leica</td>
			<td >CM1950</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection Microscope</td>
			<td >Leica</td>
			<td >M125 C</td>
			<td>Used for post-necroscopy imaging</td>
		</tr>
		<tr >
			<td >Dissolvable Sutures</td>
			<td >Med Vet International</td>
			<td >J385H</td>
			<td></td>
		</tr>
		<tr >
			<td >Distilled Water</td>
			<td >Gibco</td>
			<td >15230162</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Phosphate Buffered Saline (DPBS)</td>
			<td >Thermo fisher Scientific</td>
			<td >14190144</td>
			<td>(-) Calcium; (-) Magnesium</td>
		</tr>
		<tr >
			<td >ECM 830 Electro Electroporator (Electroporation Machine)</td>
			<td >BTX Harvard Apparatus</td>
			<td >45-0662</td>
			<td>Generator only</td>
		</tr>
		<tr >
			<td >Electric Razor</td>
			<td >Wahl</td>
			<td >CL9990</td>
			<td>Kent Scientific</td>
		</tr>
		<tr >
			<td >Electroporation paddles/Tweezertrodes</td>
			<td >BTX Harvard Apparatus</td>
			<td >45-0487</td>
			<td>3 mm diameter paddles; wires included</td>
		</tr>
		<tr >
			<td >Embedding Cassette: 250 PK</td>
			<td >Grainger</td>
			<td >21RK94</td>
			<td>Placenta embedding cassettes</td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >Thermo Fisher Scientific</td>
			<td >268280010</td>
			<td></td>
		</tr>
		<tr >
			<td >F-Air Canisters</td>
			<td >Penn Veterinary Supply Inc</td>
			<td >BIC80120</td>
			<td>Excess isoflurane filter</td>
		</tr>
		<tr >
			<td >Fast Green Dye FCF</td>
			<td >Sigma</td>
			<td >F7252-5G</td>
			<td>Dissolve to 1 &#956;g/ml and filter; protect from light</td>
		</tr>
		<tr >
			<td >Filter-based microplate photometer (plate reader)</td>
			<td >Fisher Scientific</td>
			<td >14377576</td>
			<td>Can be used for BCA and ELISA</td>
		</tr>
		<tr >
			<td >Forceps</td>
			<td >VWR</td>
			<td >82027-386</td>
			<td>Fine tips, straight, serrated</td>
		</tr>
		<tr >
			<td >Formalin solution, neutral buffered, 10%</td>
			<td >Sigma Aldrich</td>
			<td >HT501128</td>
			<td></td>
		</tr>
		<tr >
			<td >Glass Capillaries - Borosilicate Glass (Micropipette)</td>
			<td >Sutter Instrument</td>
			<td >B150-86-10</td>
			<td>O.D.: 1.5 mm, I.D.: 0.86 mm, 10 cm length</td>
		</tr>
		<tr >
			<td >Halt Protease and Phosphotase inhibitor cocktail (100x)</td>
			<td >Thermo Scientific</td>
			<td >1861281</td>
			<td>Protein homogenization buffer</td>
		</tr>
		<tr >
			<td >Heating Pad</td>
			<td >Thermotech</td>
			<td >S766D</td>
			<td>Digitial Moist Heating Pad</td>
		</tr>
		<tr >
			<td >Hemostats</td>
			<td >VWR</td>
			<td >10806-188</td>
			<td>Fully surrated jaw; curved</td>
		</tr>
		<tr >
			<td >Hot Water Bath</td>
			<td >Fisher Scientific</td>
			<td >20253</td>
			<td>Isotemp 205</td>
		</tr>
		<tr >
			<td >Igf-1 SAM Plasmid (m1)</td>
			<td >Santa Cruz Biotechnology</td>
			<td >sc-421056-ACT</td>
			<td>Dnase-free water provided for dilution</td>
		</tr>
		<tr >
			<td >Induction Chamber</td>
			<td >Vetamac</td>
			<td >941443</td>
			<td>No specific liter size required</td>
		</tr>
		<tr >
			<td >Isoflurane</td>
			<td >Piramal Pharma Limited</td>
			<td >NDC 66794-013-25</td>
			<td></td>
		</tr>
		<tr >
			<td >Isoproponal/2-Proponal</td>
			<td >Fisher Scientific</td>
			<td >A451-4</td>
			<td>RNA isolation</td>
		</tr>
		<tr >
			<td >Ketamine HCl 100mg/ml</td>
			<td >Akorn</td>
			<td >NDC 59399-114-10</td>
			<td></td>
		</tr>
		<tr >
			<td >MgCl2/Magneisum Chloride</td>
			<td >Sigma Aldrich</td>
			<td >63069-100ML</td>
			<td>1M. Protein homogenization buffer</td>
		</tr>
		<tr >
			<td >MicroAmp&#8482; Optical 384-Well Reaction Plate with Barcode</td>
			<td >Fisher Scientific</td>
			<td >4309849</td>
			<td>Barcoded plates not required</td>
		</tr>
		<tr >
			<td >Microcapillary Tip</td>
			<td >Eppendorf</td>
			<td >5196082001</td>
			<td>Attached to BTX Microinjector</td>
		</tr>
		<tr >
			<td >Microinjector</td>
			<td >BTX Harvard Apparatus</td>
			<td >45-0766</td>
			<td>Stainless Steel Pipette Holder, 130 mm Length, for 1 to 1.5 mm Pipettes</td>
		</tr>
		<tr >
			<td >Microject 1000A (Injection Machine)</td>
			<td >BTX Harvard Apparatus</td>
			<td >45-0751</td>
			<td>MicroJect 1000A Plus System</td>
		</tr>
		<tr >
			<td >Micropipette Puller Model P-97</td>
			<td >Sutter Instrument</td>
			<td >P-97</td>
			<td>Flaming/Brown type micropipette puller</td>
		</tr>
		<tr >
			<td >Microplate Mixer (Plate Shaker)</td>
			<td >scilogex</td>
			<td >822000049999</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse/Rat IGF-I/IGF-1 Quantikine ELISA Kit</td>
			<td >R &#38; D Systems</td>
			<td >MG100</td>
			<td></td>
		</tr>
		<tr >
			<td >Needles</td>
			<td >BD - Becton, Dickson, and Company</td>
			<td >305106</td>
			<td>30 Gx 1/2 (0.3 mm x 13 mm)</td>
		</tr>
		<tr >
			<td >Nitrogen Tank</td>
			<td >Linde</td>
			<td >7727-37-9</td>
			<td>Any innert gas</td>
		</tr>
		<tr >
			<td >Non-Steroidal Anti-Inflammatory Drug (NSAID)</td>
			<td >Norbrook Laboratories Limited</td>
			<td >NDC 55529-040-10</td>
			<td>Analesgic such as Meloxicam</td>
		</tr>
		<tr >
			<td >Nose Cone</td>
			<td >Vetamac</td>
			<td >921609</td>
			<td>9-14 mm</td>
		</tr>
		<tr >
			<td >Opal 620 detection dye</td>
			<td >Akoya Biosciences</td>
			<td >SKU FP1495001KT</td>
			<td>Used for FISH</td>
		</tr>
		<tr >
			<td >Optimal Cutting Temperature (O.C.T) Compound</td>
			<td >Sakura</td>
			<td >4583</td>
			<td></td>
		</tr>
		<tr >
			<td >Oxygen Tank</td>
			<td >Linde</td>
			<td >7782 - 44 - 7</td>
			<td>Medical grade oxygen</td>
		</tr>
		<tr >
			<td >Pestles</td>
			<td >USA Scientific Inc</td>
			<td >14155390</td>
			<td></td>
		</tr>
		<tr >
			<td >Povidone-Iodine Solution, 5%</td>
			<td >Avrio Health L.P.</td>
			<td >NDC 67618-155-16</td>
			<td></td>
		</tr>
		<tr >
			<td >Power SYBR&#8482; Green PCR Master Mix</td>
			<td >Thermo Fisher Scientific</td>
			<td >4367659</td>
			<td>Use for qPCR</td>
		</tr>
		<tr >
			<td >Random Hexamers (Random Primers)</td>
			<td >New England Biolabs</td>
			<td >S1330S</td>
			<td>Use for cDNA synthesis</td>
		</tr>
		<tr >
			<td >Razor Blade</td>
			<td >Grainger</td>
			<td >26X080</td>
			<td></td>
		</tr>
		<tr >
			<td >RNA Cleanup Kit &#38; Concentrator</td>
			<td >Zymo Research</td>
			<td >R1013</td>
			<td></td>
		</tr>
		<tr >
			<td >RNALater</td>
			<td >Thermo Fisher Scientific</td>
			<td >AM7021</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAscope kit v.2.5</td>
			<td >Advanced Cells Diagnostics</td>
			<td >323100</td>
			<td >Contains all reagents required for fluorescent in situ hybridization. Probes sold separately.</td>
		</tr>
		<tr >
			<td >RNAscope&#8482; Probe- Mm-Prl8a8-C2</td>
			<td >Advanced Cells Diagnostics</td>
			<td >&#160;528641-C2</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAscope&#8482; Probe- Vector-dCas9-3xNLS-VP64</td>
			<td >Advanced Cells Diagnostics</td>
			<td >527421</td>
			<td></td>
		</tr>
		<tr >
			<td >Roto-Therm Mini</td>
			<td >Benchmark</td>
			<td >R2020</td>
			<td>Dry oven for in situ hybridization</td>
		</tr>
		<tr >
			<td >Scissors</td>
			<td >VWR</td>
			<td >82027-578</td>
			<td>Dissecting Scissors, Sharp Tip, 4&#185;/&#8322;</td>
		</tr>
		<tr >
			<td >Sodium Chloride (Saline)</td>
			<td >Hospra</td>
			<td >NDC 0409-4888-03</td>
			<td>Sterile,&#160; 0.9%</td>
		</tr>
		<tr >
			<td >Sodium Citrate, Trisodium Salt, Dihydrate, [Citric Acid, Trisodium Dihydrate]</td>
			<td >Research Product International</td>
			<td >03-04-6132</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Hydroxide 1N Concentrate, Fisher Chemical</td>
			<td >Fisher Scientific</td>
			<td >SS277</td>
			<td>Protein homogenization buffer</td>
		</tr>
		<tr >
			<td >Steamer</td>
			<td >Bella</td>
			<td >B00DPX8UBA</td>
			<td></td>
		</tr>
		<tr >
			<td >Sterile Surgical Drape</td>
			<td >Busse</td>
			<td >696</td>
			<td>Sterilize before use</td>
		</tr>
		<tr >
			<td >Superfrost Plus Microscope Slides</td>
			<td >Fisher Scientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >Surgipath Cover Glass 24x60</td>
			<td >Leica</td>
			<td >3800160</td>
			<td></td>
		</tr>
		<tr >
			<td >Syringes</td>
			<td >BD - Becton, Dickson, and Company</td>
			<td >309659</td>
			<td>BD Luer Slip Tip Syringe sterile, single use, 1 mL</td>
		</tr>
		<tr >
			<td >Thermo Scientific&#8482; Invitrogen&#8482; Nanodrop&#8482; One Spectrophotometer with WiFi and Qubit&#8482; 4 Fluorometer</td>
			<td >Fisher Scientific</td>
			<td >13-400-525</td>
			<td>This configuration comes with Qubit 4 fluorometer.&#160; Qubit quantification not required.</td>
		</tr>
		<tr >
			<td >Tissue Adhesive</td>
			<td >3M</td>
			<td >1469SB</td>
			<td>VetBond</td>
		</tr>
		<tr >
			<td >Tris HCl</td>
			<td >Thermo Fisher Scientific</td>
			<td >15568025</td>
			<td>1M. Protein homogenization buffer</td>
		</tr>
		<tr >
			<td >TRIzol&#8482; Reagent</td>
			<td >Thermo Fisher Scientific</td>
			<td >15596018</td>
			<td>RNA isolation</td>
		</tr>
		<tr >
			<td >TSA Buffer Pack</td>
			<td >Advanced Cells Diagnostics</td>
			<td >322810</td>
			<td>Used to dilute Opal 620 detection dye</td>
		</tr>
		<tr >
			<td >Universal F-Circuit</td>
			<td >Vetamac</td>
			<td >40200</td>
			<td>Attached to vaporizer and vaporizer accessories</td>
		</tr>
		<tr >
			<td >Upright Compound Fluorescence Microscope</td>
			<td >Olympus</td>
			<td >BX61VS</td>
			<td>Used for FISH imaging</td>
		</tr>
		<tr >
			<td >Vectorshield with DAPI</td>
			<td >Vector Laboratories</td>
			<td >H-1200</td>
			<td>Coverslip mounting media</td>
		</tr>
		<tr >
			<td >ViiA&#8482; 7 Real-Time PCR System with 384-Well Block</td>
			<td >Thermo Fisher Scientific</td>
			<td >4453536</td>
			<td>This is for SYBR 384-well block detection.&#160; TaqMan and/or smaller blocks available</td>
		</tr>
		<tr >
			<td >Wet n Wild Nail Polish Wild Shine, Clear Nail Protector, Nail Color</td>
			<td >Amazon</td>
			<td >C450B</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylazine 20mg/ml</td>
			<td >Anased</td>
			<td >343730_RX</td>
			<td></td>
		</tr>
	</tbody>
</table>

mouse in vivo placental targeted crispr manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients and waste between the mother and fetus and the production and delivery of growth factors and hormones. Placental genetic manipulations in mice are critical for understanding the placenta's specific role in prenatal development. Placental-specific Cre-expressing transgenic mice have varying effectiveness, and other methods for placental gene manipulation can be useful alternatives. This paper describes a technique to directly alter placental gene expression using CRISPR gene manipulation, which can be used to modify the expression of targeted genes. Using a relatively advanced surgical approach, pregnant dams undergo a laparotomy on embryonic day 12.5 (E12.5), and a CRISPR plasmid is delivered by a glass micropipette into the individual placentas. The plasmid is immediately electroporated after each injection. After dam recovery, the placentas and embryos can continue development until assessment at a later time point. The evaluation of the placenta and offspring after the use of this technique can determine the role of time-specific placental function in development. This type of manipulation will allow for a better understanding of how placental genetics and function impact fetal growth and development in multiple disease contexts.

General procedure outcomes (Figure 6) 
In the study, there were three manipulated groups. These included placentas injected with a general CRISPR Cas9 control plasmid (Cas9 Control), an activation control CRISPR plasmid (Act Control), or an IGF-1 SAM activation plasmid (Igf1-OE). The Cas9 Control is better suited for knockout plasmids, and the activation control is better suited for overexpression/activation plasmids. To assess the viability ch...

Watch this Scientific Journal Video about Mouse In Vivo Placental Targeted CRISPR Manipulation at JoVE.com

Mouse In Vivo Placental Targeted CRISPR Manipulation

Here we describe a time-specific method to effectively manipulate critical developmental pathways in the mouse placenta in vivo. This is performed through the injection and electroporation of CRISPR plasmids into the placentas of pregnant dams on embryonic day 12.5.

Mouse In Vivo Placental Targeted CRISPR Manipulation

The placenta is an essential organ that regulates and maintains mammalian development in utero. The placenta is responsible for the transfer of nutrients ...

Research

JoVE Journal

Genetics

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast

We describe a PCR- and homologous recombination-based system for generating targeted mutations in histone genes in budding yeast cells. The resulting ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Manipulation of Ploidy in Caenorhabditis elegans

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or ...

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

The clustered regularly interspaced short palindromic repeats (CRISPR) system functions naturally in bacterial adaptive immunity, but has been ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...