Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation

Abstract

The ability to manipulate gene expression is the cornerstone of modern day experimental embryology, leading to the elucidation of multiple developmental pathways. Several powerful and well established transgenic technologies are available to manipulate gene expression levels in mouse, allowing for the generation of both loss- and gain-of-function models. However, the generation of mouse transgenics is both costly and time consuming. Alternative methods of gene manipulation have therefore been widely sought. In utero electroporation is a method of gene delivery into live mouse embryos^1,2 that we have successfully adapted^3,4. It is largely based on the success of in ovo electroporation technologies that are commonly used in chick⁵. Briefly, DNA is injected into the open ventricles of the developing brain and the application of an electrical current causes the formation of transient pores in cell membranes, allowing for the uptake of DNA into the cell. In our hands, embryos can be efficiently electroporated as early as embryonic day (E) 11.5, while the targeting of younger embryos would require an ultrasound-guided microinjection protocol, as previously described⁶. Conversely, E15.5 is the latest stage we can easily electroporate, due to the onset of parietal and frontal bone differentiation, which hampers microinjection into the brain. In contrast, the retina is accessible through the end of embryogenesis. Embryos can be collected at any time point throughout the embryonic or early postnatal period. Injection of a reporter construct facilitates the identification of transfected cells.

To date, in utero electroporation has been most widely used for the analysis of neocortical development^1,2,3,4. More recent studies have targeted the embryonic retina^7,8,9 and thalamus^10,11,12. Here, we present a modified in utero electroporation protocol that can be easily adapted to target different domains of the embryonic CNS. We provide evidence that by using this technique, we can target the embryonic telencephalon, diencephalon and retina. Representative results are presented, first showing the use of this technique to introduce DNA expression constructs into the lateral ventricles, allowing us to monitor progenitor maturation, differentiation and migration in the embryonic telencephalon. We also show that this technique can be used to target DNA to the diencephalic territories surrounding the 3^rd ventricle, allowing the migratory routes of differentiating neurons into diencephalic nuclei to be monitored. Finally, we show that the use of micromanipulators allows us to accurately introduce DNA constructs into small target areas, including the subretinal space, allowing us to analyse the effects of manipulating gene expression on retinal development.

Discussion

In utero electroporation can be used to analyze a wide variety of developmental processes. For example, transfection of reporter genes such as GFP, mCherry or alkaline phosphatase can be used to conduct lineage tracing and neuronal migration experiments. Alternatively, Cre recombinase can be transiently expressed to selectively eliminate a floxed allele in a spatially- and/or temporally-controlled manner. Furthermore, shRNA or dominant negative constructs can be electroporated to knockdown target gene function. Finally, targeted overexpression or misexpression of key genes in both wild type and/or genetically mutant mouse lines can be used to study cell fate decisions. The high throughput of this assay is critical as it allows for the testing of many combinations of factors in a very short time. One note of caution is that this procedure does cause changes in gene expression in cells that line the needle entry site (i.e., injury-response genes upregulated in wound; as observed by us and others¹³). It is thus recommended to focus on electroporated cells outside of the wound site. In addition, embryonic survival rates are low when first learning this technique but quickly rise to >95% with practice. To date, we have successfully used in utero electroporation technologies to identify genes regulated by proneural bHLH transcription factors in the telencephalon⁴. We have also validated the use of this technique for the analysis of telencephalic cis-regulatory elements³.

Disclosures

No conflicts of interest declared.

Acknowledgements

The authors would like to thank Eva Hadzimova, Pierre Mattar and Christopher Kovach for their initial work in establishing in utero electroporation technology in the CS lab. This work was funded by a Canadian Institute of Health Research (CIHR) grant (MOP 44094) and CIHR/Foundation Fighting Blindness (FFB) Emerging Team Grant (00933-000) to CS and an Alberta Children’s Hospital Research Foundation Grant to DMK. RD was supported by a CIHR Canada Hope Scholarship, RC is supported by an FFB Studentship and LML was supported by a CIHR Training Grant in Genetics and Child Development.

Materials

Name of reagent	Company	Catalogue Number	Category
Fine scissors	Fine Science Tools Inc.	14078-10	Surgical Tools
Iris scissors, curved	Fine Science Tools Inc.	14061-10	Surgical Tools
Olsen-Hegar Ex-Delicate Needle Holder	Fine Science Tools Inc.	12002-12	Surgical Tools
Ring forceps, 9mm	Fine Science Tools Inc.	11103-09	Surgical Tools
Eye dressing Forcep	Fine Science Tools Inc.	11051-10	Surgical Tools
Dumont #7 DMX Forcep	Fine Science Tools Inc.	11271-30	Surgical Tools
Dumont #5 DMX Forcep	Fine Science Tools Inc.	11251-30	Surgical Tools
Tissue forcep-Adson	Fine Science Tools Inc.	11027-12	Surgical Tools
Reflex Clip Applier	World Precision Instrument	500343	Surgical Tools
Perforated Spoon, 15 mm diameter	Fine Science Tools Inc.	10370-18	Surgical Tools
Autoclip Remover	Mikron	427637	Surgical Tools
Silk Black Braided Suture	Ethicon Inc.	K871	Surgical Tools
Reflex Skin Closure Stainless Steel Wound Clips	World Precision Instruments	500346	Surgical Tools
ECM 830 Square Wave Electroporation System	VWR-CanLab	58018-004	Instruments
Tweezers w/Variable Gap 2 Round 5mm Platinum Plate Electrode	Protech International Inc.	CUY650P5	Instruments
Tweezers w/Variable Gap 2 Round 7mm Platinum Plate Electrode	Protech International Inc.	CUY650P7	Instruments
Eppendorf Femtojet Microinjector	VWR CanLab	CA62111-488	Instruments
Foot Control for Eppendorf Femtojet Microinjector	VWR CanLab	CAACCESS (misc.)	Instruments
Bransonic Ultrasonic Cleaner Model 1510R-DTH	VWR CanLab	CA33995-534 CPN-952-118	Instruments
Sutter P97 Micropipet Puller	Sutter Instrument, Carsen Group Inc.	P-97	Instruments
Micropipettes – Borosilicate with filament O.D.: 1mm, I.D.: 0.78 mm, 10 cm length	Sutter Instrument	BF100-78-10	Instruments
3-Axis Coarse Manipulator	Carl Zeiss Canada Inc.	M-152	Instruments
Magnetic Holding Device for micromanipulator	World Precision Instruments	M1	Instruments
Steel Base Plate for micromanipulator	World Precision Instruments	5052	Instruments
Micropipette Holder	World Precision Instruments	MPH3	Instruments
Micropipette Handle	World Precision Instruments	5444	Instruments
Stereomicroscope	Leica	MZ6	Instruments
Vaporizer for isoflurane anesthetic	Porter Instruments Company	MODEL 100-F	Instruments
Metriclean2 Low foaming solution for sonicating surgical tools	Metrex Research Corporation	10-8100	Surgical Reagents
Gentamicin 40mg/ml in 0.2 g methylene blue antibiotic spray after suturing	Sigma Aldrich	G1264	Surgical Reagents
Germex for sterilizing surgical tools	Vétoquinol	DIN# 00141569	Surgical Reagents
BNP ophthalmic ointment	Vétoquinol	DIN# 00516414	Surgical Reagents
Nair®	Distributed by Church & Dwight Co., Inc.	commercially available	Surgical Reagents
Stanhexidine 4% w/v skin cleaner	Omega Laboratories Inc.	01938983	Surgical Reagents
Buprenorphine (Temgesic) analgesic	Schering-Plough	531-535	Surgical Reagents
Sulpha “25” sulphamethazine oral antibiotic	Professional Veterinary Laboratories	DIN# 00308218	Surgical Reagents
Lactated Ringer Solution	Baxter Corporation	DIN# 0061085	Surgical Reagents
Saline – 0.9% sodium chloride	B-Braun Medical Inc.	DIN# 01924303	Surgical Reagents
Inhalation Anesthetic – Isoflurane USP	Pharmaceutical Partners of Canada Inc.	DIN# 02237518	Surgical Reagents
Fast Green FCF	Sigma-Aldrich	F7252	Surgical Reagents

References

1. Saito, T., & Nakatsuji, N. Efficient gene transfer into the embryonic mouse brain using in vivo electroporation. Dev Biol 240, 237-246, doi:10.1006/dbio.2001.0439 S0012-1606(01)90439-7 [pii] (2001).
2. Takahashi, M., Sato, K., Nomura, T., & Osumi, N. Manipulating gene expressions by electroporation in the developing brain of mammalian embryos. Differentiation 70, 155-162, doi:S0301-4681(09)60445-X [pii] 10.1046/j.1432-0436.2002.700405.x (2002).
3. Langevin, L. M. et al. Validating in utero electroporation for the rapid analysis of gene regulatory elements in the murine telencephalon. Dev Dyn 236, 1273-1286, doi:10.1002/dvdy.21126 (2007).
4. Mattar, P. et al. Basic helix-loop-helix transcription factors cooperate to specify a cortical projection neuron identity. Mol Cell Biol 28, 1456-1469, doi:MCB.01510-07 [pii] 10.1128/MCB.01510-07 (2008).
5. Nakamura, H., Katahira, T., Sato, T., Watanabe, Y., & Funahashi, J. Gain- and loss-of-function in chick embryos by electroporation. Mech Dev 121, 1137-1143, doi:10.1016/j.mod.2004.05.013 S092547730400139X [pii] (2004).
6. Gaiano, N., Kohtz, J. D., Turnbull, D. H., & Fishell, G. A method for rapid gain-of-function studies in the mouse embryonic nervous system. Nat Neurosci 2, 812-819, doi:10.1038/12186 (1999).
7. Matsuda, T., & Cepko, C. L. Electroporation and RNA interference in the rodent retina in vivo and in vitro. Proc Natl Acad Sci U S A 101, 16-22, doi:10.1073/pnas.2235688100 2235688100 [pii] (2004).
8. Petros, T. J., Rebsam, A., & Mason, C. A. In utero and ex vivo electroporation for gene expression in mouse retinal ganglion cells. J Vis Exp, doi:1333 [pii] 10.3791/1333 (2009).
9. Garcia-Frigola, C., Carreres, M. I., Vegar, C., & Herrera, E. Gene delivery into mouse retinal ganglion cells by in utero electroporation. BMC Dev Biol 7, 103, doi:1471-213X-7-103 [pii] 10.1186/1471-213X-7-103 (2007).
10. Kataoka, A., & Shimogori, T. Fgf8 controls regional identity in the developing thalamus. Development 135, 2873-2881, doi:dev.021618 [pii] 10.1242/dev.021618 (2008).
11. Vue, T. Y. et al. Sonic hedgehog signaling controls thalamic progenitor identity and nuclei specification in mice. J Neurosci 29, 4484-4497, doi:29/14/4484 [pii] 10.1523/JNEUROSCI.0656-09.2009 (2009).
12. Tsuchiya, R., Takahashi, K., Liu, F.C., & Takahashi, H. Aberrant axonal projections from mammillary bodies in Pax6 mutant mice: possible roles of Netrin-1 and Slit 2 in mammillary projections. J Neurosci Res 87, 1620-1633, doi:10.1002/jnr.21966 (2009).
13. Buffo, A. et al. Expression pattern of the transcription factor Olig2 in response to brain injuries: implications for neuronal repair. Proc Natl Acad Sci U S A 102, 18183-18188, doi:0506535102 [pii] 10.1073/pnas.0506535102 (2005).

Cite this Article

Dixit, R., Lu, F., Cantrup, R., Gruenig, N., Langevin, L. M., Kurrasch, D. M., Schuurmans, C., Efficient Gene Delivery into Multiple CNS Territories Using In Utero Electroporation. http://www.jove.com/details.php?id=2957 doi: 10.3791/2957. J Vis Exp. 52 (2011).