Cortex-, Hippocampus-, Thalamus-, Hypothalamus-, Lateral Septal Nucleus- and Striatum-specific In Utero Electroporation in the C57BL/6 Mouse

Summary

This protocol describes in detail how to specifically transfect different regions in the C57BL/6 central nervous system via in utero electroporation. Included in this protocol are detailed instructions for transfections of regions that develop into the cortex, hippocampus, thalamus, hypothalamus, lateral septal nucleus and striatum.

Abstract

In utero electroporation is a widely used technique for fast and efficient spatiotemporal manipulation of various genes in the rodent central nervous system. Overexpression of desired genes is just as possible as shRNA mediated loss-of-function studies. Therefore it offers a wide range of applications. The feasibility to target particular cells in a distinct area further increases the range of potential applications of this very useful method. For efficiently targeting specific regions knowledge about the subtleties, such as the embryonic stage, the voltage to apply and most importantly the position of the electrodes, is indispensable.

Here, we provide a detailed protocol that allows for specific and efficient in utero electroporation of several regions of the C57BL/6 mouse central nervous system. In particular it is shown how to transfect regions the develop into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. For this information about the appropriate embryonic stage, the appropriate voltage for the corresponding embryonic stage is provided. Most importantly an angle-map, which indicates the appropriate position of the positive pole, is depicted. This standardized protocol helps to facilitate efficient in utero electroporation, which might also lead to a reduced number of animals.

Introduction

Since the first description in 2001 by three independent groups ^1-3 in utero electroporation has become a widely used standard tool for analyzing gene expression in the rodent central nervous system. Compared to the generation of knockout mice, which is, despite continuously improving techniques, still time and money consuming, the in utero electroporation appeals due to its simplicity. So, in utero electroporation enables fast and efficient gain- and loss-of-function studies ⁴.

To transfect the cerebral regions, the solution containing the negatively charged plasmid is injected into a ventricle. During the electric pulse, the negatively charged DNA migrates towards the positive pole and therefore the transfected region can be selected simply by altering the position of the positive pole. It has frequently been shown that numerous regions of the central nervous system can be targeted ^3,5-8. For instance, recent studies show specific transfections of the hippocampus, the piriform cortex or the striatum ^9-11. However, the information about the appropriate positions are often only scarcely standardized and are not always easy to transfer to different mouse strains.

Transfection of certain embryonic stages is far from trivial. Many influencing factors must be taken into consideration when choosing the set-up for specific in utero electroporation. First, to optimally transfect the respective embryonic stages, knowledge about the appropriate voltages is needed. High voltages decrease the survival rate, whereas low voltages reduce the transfection efficiency ^2,3,12. Also the size of the electrode paddle plays a crucial role, because the use of electrode paddles that are too large results in reduced specificity or can cause death due to affection of the heart rhythm ^4,12,13. The applied voltage and the size and the position of the electrode paddle are the most important features to consider, but there are also further factors influencing the outcome of the electroporation, like the applied amount of DNA-solution.

We have developed a detailed protocol which enables fast and efficient transfection of various cerebral regions of the C57BL/6 mouse ¹². In this protocol detailed information about the voltages to be used and the size of the electrode paddle for enhanced specificity is provided. Further, information about the ventricle to be filled along with recommendations for the amount of plasmid solution and the position of the electrode is supplied. The indication of the detailed position information in a map and the further visualization of these positions enables straightforward specific and efficient in utero electroporation of the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus.

Protocol

Ethics Statement: The handling of the mice and the experimental procedures were conducted in accordance with European, national and institutional guidelines for animal care.

1. In Utero Electroporation

Note: In utero electroporation was performed as previously published^12,14. Therefore, the method is only described briefly in the following (Figure 1).

1. Preparations
   1. Prepare Fast Green colored endotoxin-free advanced transfection-grade plasmid solution (containing 1,5 pCAGGS) as previously described¹².
   2. Pull and grind (35°angle) borosilicate glass capillaries (0.8-0.9 diameter) for injections.
   3. Sterilize instruments.
2. Surgery
   1. Administer analgesics (carprofen, 4 mg/kg bodyweight, s.c., 24 hr depot) at least 30 min before starting the surgery.
   2. Keep the length of the anesthesia to a maximum of 35-45 min (Stage III - stage of surgical anesthesia according to Guedel¹⁵ - maximal 25-30 min).
   3. Anesthetize timed pregnant mice with isoflurane (induction in a chamber: 2.8%, surgery via mask: 2.5%). Confirm anesthesia by unresponsiveness to toe-pinch.
   4. Apply eye ointment to prevent eye-dryness during anesthesia.
   5. Sterilize the surgical area with 7.5% Providone-Iodine solution and cover the mouse with sterile gauze (moist with 0.9% benzyl alcohol saline solution) with only the surgical area exposed.
   6. Cut open the abdominal cavity with a scalpel (skin incision: 1.5-2 cm, muscle incision: 1-1.5 cm).
   7. Preserve the opened abdominal cavity from drying out by moistening with warmed 0.9% benzyl alcohol saline solution or other sterile isotonic solution as directed by veterinary staff or committee. 
   8. Carefully extract uterine horns (using ring forceps, do not touch the uterus with fingers).
3. Injection of DNA and electroporation. Note: The exact details for targeting specific regions are indicated in section 2.
   1. Slowly inject the colored DNA solution into the ventricle as indicated in Figure 1 and section 2 via the prepared borosilicate capillaries.
   2. Apply the appropriate voltage for the corresponding embryonic stage (e.g., 36-38 V for E14) via specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec). Pulse the electroporation paddles during the application of the voltage to minimize damage of the uterine wall.
4. Post electroporation/ post-operative care
   1. Carefully replace the uterine horns in the abdominal cavity.
   2. Suture the muscle and the skin incision separately.
   3. Let the mouse rest on a thermal support device for recovery.
   4. Supervise the mouse during recovery (behavior, feed and water consumption), 4 hr, 24 hr and 48 hr after surgery. If necessary, apply additional analgesics (carprofen, 4 mg/kg bodyweight, 24 hr depot) for further 2 days.

2. Transfection of Specific Cerebral Areas

Note: The position of the positive pole is shown as the angle between the vertical centerline through the ventricle filled with the DNA solution (the right or third ventricle) and the position of the positive pole. When filling the left ventricle the positions are mirror-inverted. In Figure 4 the positions of the ear primordial are shown, which provide important reference points.

1. Transfection of cortical areas (Figure 2A, Table 1).
   1. Perform surgery as described in section 1.2. For the in utero electroporation of cortical areas use E14 embryos.
   2. Inject 1.5-2 µl DNA-solution containing 4 µg DNA in the right lateral ventricle via the prepared borosilicate capillaries.
      1. Carefully position the embryo by using ring forceps so that there are no visible vessels above the puncture site.
      2. Slowly inject the DNA-solution approximately 0.75 mm coronal from the lambdoidal suture and 0.5 mm lateral from the sagittal suture (Figure 3). Ensure that the insertion depth is 1.2 mm (E14, measured from the uterine wall). Check for a blue-green colouring with a sharp demarcation.
   3. Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
      1. Use a 3 or 5 mm electrode paddle.
      2. Use a voltage ranging from 36-38 V.
      3. Place the center of the electrodes just anterior of the ear primordia.
      4. To transfect the retrosplenial cortex use angles from 330-0°, to transfect the motor cortex 0-40°, to transfect the somatosensory cortex 40-95°, to transfect the piriform cortex 95-110° (Figure 2A).
   4. Perform post electroporation/ post-operative care as described in section 1.4. Sacrifice the mice and analyze the embryos 4 days after electroporation (E18) as described in section 3.
2. Transfection of the hippocampus (Figure 2B, Table 1).
   1. Perform surgery as described in section 1.2. For the in utero electroporation of hippocampal formation use E15 embryos.
   2. Inject 2 µl DNA-solution containing 4 µg DNA in the right lateral ventricle via the prepared borosilicate capillaries.
   3. Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
      1. Use a 3 or 5 mm electrode paddle.
      2. Use a voltage ranging from 38-40.
      3. Place the center of the negative pole just anterior of the ear primordium and the center of the positive pole at the middle of the ear primordium.
      4. To transfect the dentate gyrus use angles from 190-210°, to transfect the cornu ammonis (CA)3 210-230°, to transfect the CA2 230-250°, to transfect the CA1 250-275° (Figure 2B).
   4. Perform post electroporation/ post-operative care as described in section 1.4. Perform analysis after complete hippocampal formation (after birth, p21) as described in section 3.
3. Transfection of the lateral septal nucleus and striatum (Figure 2C, Table 1)
   Note: The angles overlap with those for transfecting the hippocampus. To effectively transfect the lateral septal nucleus and the striatum without unwanted hippocampus transfection earlier embryonic stages (E12) have to be used.
   1. Perform surgery as described in section 1.2. For the in utero electroporation of the lateral septal nucleus and striatum use E12 embryos.
   2. Inject 1 µl DNA-solution containing 4 µg DNA in the right lateral ventricle via the prepared borosilicate capillaries.
   3. Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
      1. Use a 0.5 mm electrode.
      2. Use 33 V.
      3. Place the center of the electrodes at the level of the ear primordia.
      4. To transfect the lateral septal nucleus use angles from 100-170°, to transfect the striatum 170-240° (Figure 2C).
   4. Perform post electroporation/ post-operative care as described in section 1.4. Sacrifice the mice and analyze the embryos 6 days after electroporation (E18) as described in section 3.
4. Transfection of the thalamus and hypothalamus (Figure 2D, Table 1)
   1. Perform surgery as described in section 1.2. For the in utero electroporation of the thalamus and hypothalamus use E12-E13 embryos.
   2. Inject 1-1.5 µl DNA-solution containing 4 µg DNA in the lateral ventricle (left or right)via the prepared borosilicate capillaries. Wait for 2-3 min, until the solution is diffused into the third ventricle. Alternatively directly inject into the third ventricle.
      Note: This enhances specificity but might cause a decreased survival rate (high risk of damage).
   3. Apply voltage via the specialized forceps-type platinum electrodes (interval cycle length 50 msec, interval pause 950 msec).
      1. Use a 0.5 or 3 mm electrode.
      2. Use a voltage ranging from 33-35 V.
      3. Place the center of the electrodes just posterior the ear primordia.
      4. To transfect the thalamus use angles from 25-40°, to transfect the hypothalamus 90-180° (Figure 2D).
   4. Perform post electroporation/ post-operative care as described in section 1.4. Sacrifice the mice and analyze the embryos 6 days after electroporation (E18) as described in section 3.

3. Perfusion Fixation

1. Preparations
   1. Warm 4% paraformaldehyde (PFA) to 37 °C.
   2. Fill a 50 ml syringe with PBS (4 °C) and a second syringe with warmed PFA. Avoid bubbles.
   3. Connect the syringes to a three-way stopcock. Connect the third end to a winged infusion set (butterfly).
   4. Flush the winged infusion set with PBS.
2. Perfusion
   1. Deeply anesthetize the mice (pregnant or postnatal transfected) with subcutaneous application of ketamine hydrochloride (60 mg/ kg) and xylazine (7.5 mg/ kg).
   2. Confirm the surgical tolerance stage by unresponsiveness to toe-pinch.
   3. Open the abdominal cavity just beneath the rib cage.
   4. Carefully cut the diaphragm.
   5. Carefully open the rib cage via a cut on each site up to the collarbone.
   6. Insert the tip of the butterfly into the left heart chamber (starting from the apex of the heart).
   7. Cut the left atrial appendage.
   8. Slowly perfuse the mouse with PBS (approximately 10 ml).
   9. Switch the three-way stopcock.
   10. Perfuse the mouse with 5-10 ml 4% PFA.
   11. Decapitate the mouse using scissors and dissect the brain beginning at the foramen magnum. Perform the dissection as previously published ¹⁶.
   12. Postfixate the brain for 2 days in 4% PFA. Keep the brains at 4 °C in the dark.

4. Immunohistochemistry

1. Generate 70 µm sections (e.g., with a vibratome).
2. Optional: Enhance fluorescence with antibody staining.
   1. Block the sections for 1 hr at RT (0.1-0.2% Triton X-100, 5% normal goat serum)
   2. Incubate O/N with the appropriate antibody (anti-GFP, 1:1,000)
   3. Wash 3-5 times with 0.1 M phosphate buffer.
   4. Incubate 3-5 hr with the appropriate fluorescence-conjugated secondary antibody (Alexa Fluor 488 conjugated anti-rabbit-IgG, 1:1,000).
   5. Optional: Counterstain with 4-6-diaminodino-2-phenylindole (DAPI, 0.2 g/ ml in 0.1 M phosphate buffer) for 1 min.
   6. Analyze fluorescence with an appropriate fluorescence microscope.

Results

Figure 2, shows examples for the specific in utero electroporation of the regions developing into the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus. The results of the transfections are shown next to the recommended angle (Figure 2). For better visualization of the angles in vivo the position of the electrode (0.5...

Discussion

This protocol describes in detail how to transfect the retrosplenial cortex, the motor cortex, the somatosensory cortex, the piriform cortex, the cornu ammonis 1-3, the dentate gyrus, the striatum, the lateral septal nucleus, the thalamus and the hypothalamus of C75BL/6 mice. With all the provided information this is the first protocol, which supplies all necessary information to easily recreate transfections of these cerebral regions in the C57BL/6 mouse. Previous publications are mostly focused only on a few specific r...

Materials

Name	Company	Catalog Number	Comments
EndoFree Plasmid Maxi Kit	QIAGEN	12362
Fast Green	Roth	0301.1
pCAGGS	Addgene
borosilicate glass capillaries (0.8-0.9 mm diameter)	Wold Precision Instrument Inc.	1B100F-4
Isoflurane (Forene)	Abbott	PZN 4831850
Carprofen (Rimadyl)	Pfizer GmbH	approval number: 400684.00.00
eye ointment (Bepanthen Augen und Nasensalbe)	Bayer	PZN 01578681
0.9% benzyl alcohol 0.9% saline solution	Pharmacy of the University Medical Center Mainz
gauze (ES-Kompressen)	Hartmann	407835
sterile 5-0 Perma-Hand Silk Suture	Ethicon Johnson & Johnson	K890H
ring forceps 1/ 1.5 mm	Fine Science Tools	11101-09
ring forceps 4.8/ 6 mm	Fine Science Tools	11106-09
ring forceps 2.2/ 3 mm	Fine Science Tools	11103-09
Adson Forceps-Serrated Straight 12 cm	Fine Science Tools	1106-12
IrisScissors-Delicate Straight-Sharp/Blunt 10 cm	Fine Science Tools	14028-10
Mayo-Stille Scissors-Straight 15 cm	Fine Science Tools	14012-15
Dumont #5 Forceps-Inox	Fine Science Tools	11251-20
Castroviejo NeedleHolder-with Lock-Tungsten Carbide 14 cm	Fine Science Tools	12565-14
Elektroporator CUY21 SC	Nepa Gene Co.
FST 250 Hot Bead Sterilizer	Fine Science Tools	18000-45
Microgrinder EG-44	Narishige
P-97 Micropette Puller	Sutter Instrument Company	P-97
Platinum electrodes 650P 0.5 mm	Nepagene	CUY650P0.5
Platinum electrodes 650P 3 mm	Nepagene	CUY650P3
Platinum electrodes 650P 5 mm	Nepagene	CUY650P5
Platinum electrodes 650P 10 mm	Nepagene	CUY650P10
Anesthesia system	Rothacher-Medical GmbH	CV-30511-3 Vapor 19.3
Heating plate	Rothacher-Medical GmbH	HP-1M
Temperature Controller 220V AC	Rothacher-Medical GmbH	TCAT-2LV