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Tool Use and Care
Use clamps or other practical way to secure and support the workpiece to a stable platform. Holding the work by hand or against your body is unstable and may lead to loss of control. Do not force tool. Use the correct tool for your application. The correct tool will do the job better and safer at the rate for which it is designed. Do not use tool if switch does not turn it ON or OFF. Any tool that cannot be controlled with the switch is dangerous and must be repaired. Disconnect the plug from the power source before making any adjustments, changing accessories, or storing the tool. Such preventive safety measures reduce the risk of starting the tool accidentally. Store idle tools out of reach of children and other untrained persons. Tools are dangerous in the hands of untrained users. Maintain tools with care. Keep cutting tools sharp and clean. Properly maintained tools, with sharp cutting edges are less likely to bind and are easier to control. Any alteration or modification is a misuse and may result in a dangerous condition. Check for misalignment or binding of moving parts, breakage of parts, and any other condition that may affect the tools operation. If damaged, have the tool serviced before using. Many accidents are caused by poorly maintained tools. Develop a periodic maintenance schedule for your tool.
Stay alert, watch what you are doing and use common sense when operating a power tool. Do not use tool while tired or under the influence of drugs, alcohol, or medication. A moment of inattention while operating
Use only accessories that are recommended by the manufacturer for your model. Accessories that may be suitable for one tool, may become hazardous when used on another tool.
Tool service must be performed only by qualified repair personnel. Service or maintenance performed by unqualified personnel could result in a risk of injury. For example: internal wires may be misplaced or pinched, safety guard return springs may be improperly mounted.
When servicing a tool, use only identical replacement parts. Follow instructions in the Maintenance section of this manual. Use of unauthorized parts or failure to follow Maintenance Instructions may create a risk of electric shock or injury. Certain cleaning agents such as gasoline, carbon tetrachloride, ammonia, etc. may damage plastic parts.
Safety Rules for Rotary Tools
Accessories must be rated for at least the speed recommended on the tool warning label. Wheels and other accessories running over rated speed can fly apart and cause injury. Hold tool by insulated gripping surfaces when performing an operation where the cutting tool may contact hidden wiring or its own cord. Contact with a "live" wire will make exposed metal parts of the tool "live" and shock the operator. If cutting into existing walls or other blind areas where electrical wiring may exist is unavoidable, disconnect all fuses or circuit breakers feeding this worksite. Do not operate the flexible shaft with a sharp bend. Over bending the shaft can generate excessive heat on the jacket or hand piece. The recommended minimum is 6" radius. Always disconnect the power cord from the power source before making any adjustments or attaching any accessories. You may unexpectedly cause the tool to start leading to serious personal injury. Be aware of the switch location, when placing the tool down or when picking the tool up. You may accidentally activate the switch. Always hold the hand piece firmly in your hands during the start-up. The reaction torque of the motor, as it accelerates to full speed, can cause the shaft to twist. Always wear safety goggles and dust mask. Use only in well ventilated area. Using personal safety devices and working in safe environment reduces risk of injury. After changing the bits or making any adjustments, make sure the collet nut and any other adjustment devices are securely tightened. Loose adjustment device can unexpectedly shift, causing loss of control, loose rotating components will be violently thrown. Do not reach in the area of the spinning bit. The proximity of the spinning bit to your hand may not always be obvious. Allow brushes to run at operating speed for at least one minute before using wheel. During this time no one is to stand in front or in line with the brush. Loose bristles or wires will be discharged during the run-in time. Wire and bristle brushes must never be operated at speeds greater than 15,000/min. Direct the discharge of the spinning wire brush away from you. Small particles and tiny wire fragments may be discharged at high velocity during the cleaning action with these brushes and may become imbedded in your skin. Bristles or wires will be discharged from the brush at high speeds. Wear protective gloves and face shield with wire or bristle brushes. Apply wire or bristle brushes lightly to the work as only the tips of the wire/bristles do the work. Heavy pressure on bristles will cause the wire or bristle to become overstressed, resulting in a wiping action and will cause the bristles/wire to be discharged. Carefully handle both the tool and individual grinding wheels to avoid chipping or cracking. Install a new wheel if tool is dropped while grinding. Do not use a wheel that may be damaged. Fragments from a wheel that bursts during operation will fly away at great velocity possibly striking you or bystanders. Never use dull or damaged bits. Sharp bits must be handled with care. Damaged bits can snap during use. Dull bits require more force to push the tool, possibly causing the bit to break. Use clamps to support workpiece whenever practical. Never hold a small workpiece in one hand and the tool in the other hand while in use. Allow for sufficient space, at least 6", between your hand and the spinning bit. Round material such as dowel rods, pipes or tubing have a tendency to roll while being cut, and may cause the bit to bite or jump toward you. Clamping a small workpiece allows you to use both hands to control the tool.
Safety Rules for Rotary Tools - (cont.)
Inspect your workpiece before cutting. When cutting irregularly shaped workpieces, plan your work so it will not slip and pinch the bit and be torn from your hand. For example, if carving wood, make sure there are no nails or foreign objects in the workpiece. Nails or foreign objects can cause the bit to jump. Never start the tool when the bit is engaged in the material. The bit cutting edge may grab the material causing loss of control of the cutter. Avoid bouncing and snagging the wheel, especially when working corners, sharp edges etc. This can cause loss of control and kick-back. The direction of feed with the bit into the material when carving, routing or cutting is very important. Always feed the bit into the material in the same direction as the cutting edge is exiting from the material (which is the same direction as the chips are thrown). Feeding the tool in the wrong direction, causes the cutting edge of the bit to climb out of the work and pull the tool in the direction of this feed. If the workpiece or bit becomes jammed or bogged down, turn the tool OFF by the switch. Wait for all moving parts to stop and unplug the tool, then work to free the jammed material. If the switch to the tool is left ON the tool could restart unexpectedly causing serious personal injury. Do not leave a running tool unattended, turn power off. Only when tool comes to a complete stop it is safe to put it down. Do not grind or sand near flammable materials. Sparks from the wheel could ignite these materials. Do not touch the bit or collet after use. After use the bit and collet are too hot to be touched by bare hands. Regularly clean the tool's air vents by compressed air. Excessive accumulation of powdered metal inside the motor housing may cause electrical failures. Do not allow familiarity gained from frequent use of your rotary tool to become commonplace. Always remember that a careless fraction of a second is sufficient to inflict severe injury. Do not alter or misuse tool. Any alteration or modification is a misuse and may result in serious personal injury. This product is not intended for use as a dental drill, in human or veterinary medical applications. Serious personal injury may result. When using the steel saws, cutoff wheels, high speed cutters or tungsten carbide cutters, always have the work securely clamped. Never attempt to hold the work with one hand while using any of these accessories. The reason is that these wheels will grab if they become slightly canted in the groove, and can kickback causing loss of control resulting in serious injury. Your second hand should be used to steady and guide the hand holding the tool. When a cutoff wheel grabs, the wheel itself usually breaks. When the steel saw, high speed cutters or tungsten carbide cutter grab, it may jump from the groove and you could lose control of the tool. Some dust created by power sanding, sawing, grinding, drilling, and other construction activities contains chemicals known to cause cancer, birth defects or other reproductive harm. Some examples of these chemicals are:
Lead from lead-based paints, Crystalline silica from bricks and cement and other masonry products, and Arsenic and chromium from chemically treated lumber. Your risk from these exposures varies, depending on how often you do this type of work. To reduce your exposure to these chemicals: work in a well ventilated area, and work with approved safety equipment, such as those dust masks that are specially designed to filter out microscopic particles.
IMPORTANT: Some of the following symbols may be used on your tool. Please study them
and learn their meaning. Proper interpretation of these symbols will allow you to operate the tool better and safer. Symbol V A Hz W kg min s Name Volts Amperes Hertz Watt Kilograms Minutes Seconds Diameter n0./min 0 1, 2, 3,. I, II, III,
Designation/Explanation Voltage (potential) Current Frequency (cycles per second) Power Weight Time Time Size of drill bits, grinding wheels, etc. Rotational speed, at no load
No load speed
Revolutions or reciprocation per minute Revolutions, strokes, surface speed, orbits etc. per minute Off position Selector settings Infinitely variable selector with off Arrow Alternating current Direct current Alternating or direct current Class II construction Earthing terminal Warning symbol Ni-Cad RBRC seal Zero speed, zero torque. Speed, torque or position settings. Higher number means greater speed Speed is increasing from 0 setting Action in the direction of arrow Type or a characteristic of current Type or a characteristic of current Type or a characteristic of current Designates Double Insulated Construction tools. Grounding terminal Alerts user to warning messages Designates Ni-Cad battery recycling program This symbol designates that this tool is listed to Canadian Standards by Underwriters Laboratories. This symbol designates that this tool is listed by Underwriters Laboratories, and listed to Canadian Standards by Underwriters Laboratories.
This symbol designates that this tool is listed by Underwriters Laboratories.
This symbol designates that this tool is listed by the Canadian Standards Association.
This symbol designates that this tool complies to NOM Mexican Standards.
Functional Description and Specifications
Disconnect the plug from the power source before making any assembly, adjustments or changing accessories. Such preventive safety measures reduce the risk of starting the tool accidentally.
Rotary Tool 275T6 & 285T6
COLLET NUT COLLET
SHAFT LOCK BUTTON
SWITCH (275T6 SINGLE SPEED) (285T6 TWO SPEED)
Rotary Tool 395T6
HANGER SOFT GRIP CORD
HOUSING CAP COLLET COLLET NUT
VARIABLE SPEED SWITCH
Model number Voltage rating Amperage rating No load speed Collet capacities
275T6 120V 50 - 60Hz 1.15A n0 35,000/min 1/32, 1/16", 3/32", 1/8"
285T6 120V 50 - 60Hz HI 1.15A, LO 0.80A n0 15,000/35,000/min 1/32, 1/16", 3/32", 1/8"
395T6 120V 50 - 60Hz 1.15A n0 5,000-35,000/min 1/32, 1/16", 3/32", 1/8"
FLEXIBLE SHAFT CORE
OVERTHROW NUT ASSEMBLY
Cutting Guide 565
GUIDE INSERT DEPTH ADJUSTMENT SCREW
Grout Removal Kit 568
Right Angle Attachment 575
COLLAR COLLET COLLET NUT
Always unplug Rotary Tool before changing accessories, changing collets or servicing your Rotary Tool.
COLLET IDENTIFICATION CHART Collet sizes can be identified by the rings on the back end of collet. 1/32" Collet has one (1) ring. 1/16" Collet has two (2) rings. 3/32" Collet has three (3) rings. 1/8" Collet has no rings.
SHAFT LOCK BUTTON COLLET WRENCH TO TIGHTEN COLLET NUT
COLLET NUT To loosen, first press shaft lock button and rotate the shaft by hand until the lock engages the shaft preventing further rotation.
Do not engage lock while the Rotary Tool is running.
Step 5. Attach by screwing the collar of the flex-shaft to the rotary tool. Make sure the square end of the center core engages the square hole socket in the driver cap. Do not pull out center core to engage into driver cap. This could cause disengagement of center core from handpiece. If tool stops when shaft is bent, center core may be lodged in driver cap. Loosen shaft and remove core from driver cap. Then screw flexible shaft onto rotary tool housing again.
Reattach the flex-shaft to the rotary tool.
Assembly & Operation of Attachments - (Cont.)
Do not operate the flexible shaft with a sharp bend. This can generate excessive heat and will reduce tool and flexshaft life. The recommended minimum is 5" radius.
Contents of 225 Flex-Shaft Attachment: Qty. Description Flex-Shaft Assembly (42" long) Driver Cap
The Cutting Guide 565 (sold separately) comes completely assembled and ready to use. For use in a variety of materials up to 3/4 thick. Match the bit type to the material to be cut. Always hold the tool firmly, using slow steady pressure to make cuts. To attach, follow the four steps shown below. Important: When viewing the tool from the top, the bit rotates clockwise. Feed direction of cutting must be counter-clockwise.
#560 Drywall Cutting Bit
For use in drywall. When inserting the #560 bit into your Rotary Tool, make sure that the bit has been inserted as far as possible. When using a template (outlet box) behind the drywall, use the drywall bit #560, cutting in a counterclockwise direction.
When making freehand cuts in Drywall, example repairing a hole in drywall, use the Multipurpose bit #561,cutting in a clockwise direction. When using #561, Multipurpose Cutting Bit, start the bit into the material at a 45 degree angle and then slowly bring it to a 90 degree angle to begin the cut.
#562 Tile Cutting Bit
For use on wall tile, cement board and plaster When inserting the #562 bit into your MultiPro tool, it is very important that 1/16-1/8 of smooth shank remains visible above the collet. When using #562, Tile Cutting Bit, start the bit into the material at a 45 degree angle and then slowly bring it to a 90 degree angle to begin the cut. NOT FOR USE ON FLOOR TILE
#561 Multipurpose Cutting Bit
For use in wood, plastics, drywall, fiberglass, vinyl or aluminum siding, acoustical tile and laminates. When inserting the #561 bit into your Rotary Tool, make sure that the bit has been inserted as far as possible.
The #568 grout removal attachment comes completely assembled and ready to use. Use the #569 (1/16") bit for tiles spaced more than 1/16" apart. If your tiles are spaced more than 1/8" apart, it is recommended that you use the #570 (1/8") bit. Note: If the bit is too wide for the spacing between your tiles, you may damage your tile or the grout removal bit. Step 1: Remove the housing cap from the tool. Step 2: Insert the grout removal bit into your rotary tool. When inserting the #569 or #570 grout removal bit into your Dremel rotary tool, be sure that the bit is secure within the jaws of the collet. Use the wrench to tighten the collet nut to prevent the bit from loosening within the collet. Do not use your Dremel Chuck, #4486, with the grout removal bits.
Step 3: Screw the grout removal attachment onto the rotary tool. Step 4: Adjust the attachment and bit to the desired cutting depth. Grout Removal Attachment Cutting Depth Adjustment The Multi Slide Depth Adjustment has increment markings of 1/8" (3,2 mm). These markings are for reference only in identifying the depth of your desired cut. The multiple channels of the depth adjustment let you choose the orientation of the attachment to the tool. Be sure to securely tighten the screw within one of the multiple channel positions. To set cutting depth: Cleaning Grout: Do not remove grout more than 1/8" below the face surface of the tile. Adjust the Multi Slide Depth Adjustment and bit so that no more than 1/8" of the bit extends beyond the base of the attachment. Removing Grout to Replace A Broken Tile: Remove all of the grout surrounding the broken tile. Adjust the Multi Slide Depth Adjustment so that no more than 1/8" of the bit extends beyond the base of the attachment. See Figure 5. Remove grout at a depth no more than 1/8" at a time. You may need to adjust the Multi Slide Depth Adjustment by 1/8" increments (reference the 1/8" incremental white markings on the Multi Slide Depth Adjustment) and make several passes until all the grout is removed. When removing grout deeper into the grout line, you may strike hidden objects like screw heads, mortar, tile cement or nails that may cause the bit to bind, overheat or break. Reduce the tool speed and work through it slowly, making several passes. In case of screws or nails, remove the grout around the area as the bit will not cut through them.
After removing 1/8" of grout, regrout to tile level. Seal the new grout.
Operating Instructions Always pull the tool toward you! ! WARNING Do not push it! Pushing the bit may cause it to break. Hold the tool in a golf grip with the tool positioned below the attachment and the bit pointing upwards. On your variable speed tool, recommended tool speed is 15,000-20,000 RPM's or speed setting 6 to avoid damage to the bit. On your two speed tool, recommended tool speed is "Low" to avoid damage to the bit. Do not force the bit or put pressure on the back of the tool to remove the grout. Let the speed of the rotating bit do the work. Wear eye protection and dust mask. Inspect bit for damage. When bit is installed, always run it at no-load speed of the tool for one minute, as a damaged bit will break apart. Do not stand in front of or in line with bit.
Always use the tool with the depth guide positioned flat against the material being cut. The guide securely positioned on the material improves stability and control of your tool. The direction of feed with the bit into the grout is important. Always drag or pull the bit through the grout line. The grout bit is not intended for "plowing" through the grout and feeding the tool in the wrong direction will cause the bit to climb out of the work possibly damaging the bit and/or causing loss of control.
Before you begin, remove the black protective cap on your attachment. If cap does not slide off easily, insert the shank portion of any accessory through the housing opening of the attachment to hold shaft from rotating. Then twist off. Figure 1. Do not use the rotary tool shaft lock button when changing accessories on the attachment. Internal damage to the attachment may occur.
Screw the attachment onto your rotary tool. Hand tighten only. Reassemble the collet and the collet nut from step 2, on to the output shaft of the attachment. Figure 4. The right angle attachment can be oriented on your rotary tool in 12 different positions. The attachment should be positioned so the on/off speed control switch is easy to access. To reposition, unscrew the collar from the attachment until disengaged. Slide the attachment off. Then, reposition, slide the attachment back on the tool and retighten the collar. Figure 5. To change an accessory, insert the shank portion of any accessory (3,2 mm recommended) through the housing opening of the attachment to hold the shaft from rotating. With the shaft secured, loosen the collet nut and insert an accessory as deeply as possible to avoid wobble during use. You may need to pull back the shank from the housing opening to provide clearance while inserting the accessory. Figure 6.
225 - Flex-Shaft Allows finger-tip control for tight corners and hard-to-reach areas. 36" long cable attaches to Models 275, 285, 395, and 850. Pencillike 1/2" diameter hand piece is cool-running and ideal for light duty wood carving and other uses.
231 - Shaper/Router Table Converts the Rotary Tool into a bench mounted wood shaper. Clamp it to a workbench and perform professional quality slotting, edge trimming, grooving and sanding of irregular shapes accurately and with ease. Large 8" x 6" worktable. Use with Models: 270, 275, 280, 285, 370, 380, 395 and the 850.
212 - Drill Press For precision drilling, routing, grooving, 6" square work surface, 0" to 3" throat depth. Table slotted for guides, hold downs. Holds Models 275, 285, 395 and 850.
575 - Right Angle Attachment Enhances the versatility of your Dremel rotary tool by allowing you to get into hard-to-reach areas.
568 - Grout Removal Kit Four use on wall and floor grout 30 angle for controlled cutting Guides 180 apart to keep bit centered between tiles Easy screw on mounting
2217 - Tool Holder and Base Firmly holds rotary tools in any position. Control workpiece (hands free) for better results.
Use only Dremel Tested, High Performance Accessories. Other accessories are not designed for this tool and may lead to personal injury or property damage. The number and variety of accessories for the Rotary Tool are almost limitless. There is a category suited to almost any job you might have to do and a variety of sizes and shapes within each category which enables you to get the perfect accessory for every need. Refer to the DREMEL ACCESSORY ORDER FORM for illustrations of the accessories available. These accessories may be found at your local hardware, hobby or home center dealers.
Tungsten Carbide Cutters
These are tough, long-lived cutters for use on hardened steel, fired ceramics and other very hard materials. They can be used for engraving on tools and garden equipment. 1/8" shanks.
This group has a wide variety of sizes and shapes, and are made for intricate work on ceramics (greenware), wood carvings, jewelry and scrimshaw. They often are used in making complicated printed circuit boards. They should not be used on steel and other very hard materials but are excellent on wood, plastic and soft metals. 3/32" shank.
If you expect to use a variety of accessories, we recommend that in the beginning you purchase a complete set of four collets. Store these so that you will have the proper size of collet for any accessory or drill bit you want to use. Currently, the 1/8", 3/32",1/32" and 1/16" collets accommodate all of the available Dremel accessories. 1/8" collets are included in most rotary tool kits.
A mandrel is a shank with a threaded or screw head, which are required when you use polishing accessories, cutting wheels, sanding discs, and polishing points. The reason mandrels are used is that sanding discs, cutting wheels and similar accessories must be replaced frequently. The mandrel is a permanent shank, allowing you to replace only the worn head when necessary, thus saving the expense of replacing the shaft each time. Screw Mandrel No. 401 This is a screw mandrel used with the felt polishing tip and felt polishing wheels. 1/8" shank. Small Screw Mandrel No. 402 This is a mandrel with a small screw at its tip, and is used with emery and fiberglass cutting wheels, sanding discs and polishing wheels. 1/8" shank. Threaded Tip Mandrel No. 424 This is a mandrel with a threaded tip which threads into the polishing point accessory No. 427. 1/8" shank.
These include an impregnated polishing point and an impregnated polishing wheel for bringing metal surfaces to smooth finish; a felt polishing tip and felt polishing wheel, and cloth polishing wheel, all used for polishing plastics, metals, jewelry and small parts. Also included in this group is a polishing compound (No. 421) for use with the felt and cloth polishers. Polishing points make a very smooth surface, but a high luster is obtained using felt or cloth wheels and polishing compound. For best results polishing accessories should be used at speeds not greater than 15,000 RPM. Refer to Operating Speeds section for proper tool speed setting. No polishing compound is needed when using the 425 Polishing Wheel or 427 Polishing point.
These are excellent cleaning tools on silverware, jewelry and antiques. The three shapes make it possible to get into tight corners and other difficult places. Bristle brushes can be used with polishing compound for faster cleaning or polishing.
Aluminum Oxide Abrasive Wheels Brushing Pressure
1. Remember, the tips of a wire brush do the work. Operate the brush with the lightest pressure so only the tips of the wire come in contact with the work. 2. If heavier pressures are used, the wires will be overstressed, resulting in a wiping action; and if this is continued, the life of the brush will be shortened due to wire fatigue. 3. Apply the brush to the work in such a way that as much of the brush face as possible is in full contact with the work. Applying the side or edge of the brush to the work will result in wire breakage and shortened brush life.
CORRECT: Wire tips doing the work.
Use to remove paint, deburr metal, polish stainless steel and other metals. Available in fine and medium grits. 1/8" shank.
Sanding discs in fine, medium and coarse grades are made to fit mandrel No. 402. They can be used for nearly any small sanding job you might have, from model making to fine furniture finishing. In addition, there is the drum sander, a tiny drum which fits into the Rotary Tool and makes it possible to shape wood, smooth fiberglass, sand inside curves and other difficult places, and other sanding jobs. You replace the sanding bands on the drum as they become worn and lose their grit. Bands come in fine and coarse grades. Flapwheels grind and polish flat or contoured surfaces. They are used most effectively as a finishing sander after heavier surface sanding and material removal is completed. Flapwheels come in fine and coarse grades. Buffs are a great finishing accessory for cleaning and light sanding. They work effectively on metal, glass, wood, aluminum and plastics. Coarse and medium buffs are sold together. 1/8" shank.
INCORRECT: Excessive pressure can cause wire breakage.
Use for deburring, removing rust, and general purpose grinding. Use with Mandrel #402.
Dremel Accessories - (Cont.)
Tile Cutting Bit
Cuts ceramic wall tile, cement board, and plaster.
These thin discs of emery or fiberglass are used for slicing, cutting off and similar operations. Use them for cutting off frozen bolt heads and nuts, or to reslot a screw head which has become so damaged that the screwdriver wont work in it. Fine for cutting BX cable, small rods, tubing, cable and cutting rectangular holes in sheet metal.
Spiral Cutting Bit
Cuts through all types of wood and wood composites.
Drywall Cutting Bit
Gives you fast, clean cuts in drywall.
High Speed Router Bits For routing, inlaying, and mortising in wood and other soft materials. Use only with Dremel No. 330 Router attachment or No. 231 Shaper/Router table.
Mandrel No. 401 is used with the felt polishing tip and wheels. Thread the tip on to the screw carefully. The felt tip must thread down straight on the screw Mandrel, and be turned all the way to the collar.
Mandrel No. 402 has a small screw at its tip, and is used with emery cutting wheels and sanding discs. Higher speeds, usually maximum, are best for most work, including cutting steel. Which is shown here.
The machine-screw threading on Mandrel No. 424 threads into polishing point No. 427. This and other threaded mandrels must be screwed firmly down to the collar before being used.
To replace a band on the Drum Sander, loosen the screw without removing it to contract the drum then slide the old band off. Slide the new sanding band on and then expand the drum by tightening the screw once again. Before each use, check to make certain that all components are assembled to accessory shank and that the drum is sufficiently expanded to secure the band during use. If sanding band is loose on the drum during operation it may fly off and strike you or bystanders.
SOFT WOOD STEEL CERAMIC HARD WOOD LAMINATES PLASTIC ALUMINUM, BRASS, ETC. SHELL/ STONE GLASS
* Speed for light cuts, caution burning on deep grooves. Depending on cutting direction relative to grain.
HIGH SPEED CUTTERS
100, 121, 131 114, 124, 134, 118, 191, 192, 193, 194 116, 117, 125, 6 6
10 10* 10* 10* 6 8* 6* 6* 10 10* 10* 10*
105, 108 106, 109 107, 112 113
DIAMOND WHEEL POINTS
7103, 7105, 7117, 7120, 7122, 7123, 7134, 7144
STRUCTURED TOOTH TUNGSTEN CARBIDE CUTTERS
9931, 9932, 9933, 9934, 9935, 9936
TUNGSTEN CARBIDE CUTTERS
8 8-10 8-10 8-10 8-10
9901, 9902, 9903, 9904, 9905, 9906, 9912
9909, 9910, 9911
SOFT WOOD LAMINATES PLASTIC STEEL ALUMINUM, BRASS, ETC. SHELL/ STONE CERAMIC HARD WOOD
HIGH SPEED ROUTER BITS
10* 10* 10* 8
612, 640 615, 617, 618, 650, 652 654
SILICON CARBIDE GRINDING STONES
83142, 83322, 83702, 84922, 85422, 85602, 85622
4-6 4-6-8 4-6 4-4-6
516, 517, 518 500
ALUMINUM OXIDE GRINDING STONES
903, 911, 921, 932, 941, 945, 952, 953, 954, 971, 997, 8153, 8175, 8193, 8215 541
CHAIN SAW SHARPENING STONES CUTTING ACCESSORIES
10 6-6-8 2-4 2-4 8-8 8-10 For use on drywall. For best results, use at 30,000 rpm. 2-4 6-10 6-10
453, 454, 455
409, 420, 426, 561 562
SOFT WOOD STEEL CERAMIC HARD WOOD LAMINATES PLASTIC SHELL/ STONE 6-8 6-6 ALUMINUM, BRASS, ETC.
4 2-10 2-4 8-2-10 2-10 2-4
461, 462, 463 414, 422, 429 425, 403, 404, 405 530, 531, 532 428, 442, 443 535, 536, 6-8 6
8 6-10 10
SANDING BANDS AND DISCS
2-10 2-10 2-2-10 2-10 2-2-6 2-6 2-6 2-4 2-10 2-10 2-10
430, 431, 438 439, 440, 444 407, 408, 432 411, 412, 413
502, 503, 504, 505 511
FINISHING ABRASIVE BUFFS DRILL BIT
GROUT REMOVAL BITS
For Use on Wall and Floor Grout 6-8
MODELS MODLES MODELOS 275T6, 285T6, 395T6
ORDER BY PART NUMBER, NOT CODE NUMBER COMMANDEZ PAR LE NUMRO DE LA PICENON PAR LE NUMRO DE CODE ORDERNE POR NUMERO DE PIEZA, NO POR NUMERO DE CODIGO
CODE NO. NO. DE CODE NUMERO DE CODIGO 3 4
4/28/03 10:53 AM
PART NO. NO. DE LA PIECE NUMERO DE PIEZA *DESCRIPTION Wrench Collet Nut 1/8" Collet (In Tool) Housing Cap 3/32" Collet Armature & Bearing Assembly Field Assembly Collet Lock & Spring Housing Set Hanger Brush Cap (Pair) Brush Spring (Pair) Switch Assembly Cord Screw (individual) Isolator DESCRIPTION Cl Ecrou De Douille Douille 3,2 mm (avec l'outil) Chapeau du bti Douille 2,4 mm Ensemble de l'armature et palier Champ d'induit Verrou et ressort pour douille Bti Bride de suspension Chapeau de balai (paire) Ressort de balai (paire) Montage de l'interrupteur Cordon Vis Manchon isolant
Chapeau de la pice manuelle Ncleo del eje flexible Llave de tuerca disponible como accesorio (no incluida) Ensemble d'crou de renversement Tapa del impulsor Guia Guia para insertar Destornillador, Muelle & Tuerca
GROUT REMOVAL KIT KIT POUR LEXTRACTION DE COULIS JUEGO PARA QUITAR LECHADA
CODE NO. PART NO. NUMERO DE CODIGO NUMERO DE PIEZA NO. DE CODE NO. DE LA PIECE DESCRIPTION Angled Cone Housing Multi Channel Slide Thumbscrew Square Nut Compression Spring DESCRIPCION 2615302690
DESCRIPTION Enveloppe angulaire conique Glissire cannelures Vis ailette crou carr Ressort
Cubierta Protectora en Forma de Angulos Deslizamiento Multi-Canales Tornillo de Apriete Manual Tuerca Cuadrada Resorte
Dremel Limited Warranty
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Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo
Ayumu Tashiro1,2, Chunmei Zhao1 & Fred H Gage1
1Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037, USA. 2Present address: Centre for the Biology
of Memory, Norwegian University of Science and Technology, Medical-Technical Research Centre, NO-7489 Trondheim, Norway. Correspondence should be addressed to F.H.G. (email@example.com) Published online 25 Janaury 2007; doi:10.1038/nprot.2006.473
2006 Nature Publishing Group http://www.nature.com/natureprotocols
Single-cell genetic manipulation in an intact brain environment is an informative approach to study molecular mechanism of adult neurogenesis. Here, we describe a protocol for retrovirus-mediated single-cell gene knockout in adult new neurons in vivo. A gene of interest is disrupted in adult oxed mice by a vector based on the Moloney murine leukemia retrovirus, expressing Cre recombinase. High-titer retrovirus is prepared by transfecting plasmids into the HEK293T cells and by concentrating the supernatant containing virus. The retrovirus is stereotaxically injected into the dentate gyrus. Cre recombinase is transduced and expressed in a small fraction of adult new neurons in an intact environment, and the gene knockout is highly efcient within the transduced neurons. Virus preparation takes 7 days, but virus injections take less than 1 h per mouse. By changing the survival time of the mice after the injection, one can analyze the effects on new neurons at different ages.
INTRODUCTION In the adult mammalian brain, new neurons are generated and incorporated into the existing neural circuits in restricted regions including the dentate gyrus and the olfactory bulb1. Although studies have revealed a large body of information on its molecular mechanisms, an overall picture of how adult neurogenesis is regulated is still largely unclear2. Most of these studies have utilized global manipulation such as drug/virus infusion and conventional genetically modied mice. Although these approaches can indicate that a gene or protein of interest is somehow related to adult neurogenesis, interpretation of results is inevitably confounded by possible global alteration of the surrounding environment, for example, observed effects could be side effects caused by disturbance in the activity pattern of neighboring neural circuits or signaling from other neurons. Therefore, it is informative to examine the behavior of single neurons when a small number of neurons are manipulated leaving their surrounding environment intact. To achieve this, we developed a retrovirus-mediated single-cell knockout technique in adult new neurons in vivo3. The principle of the technique is to deliver Cre recombinase4 selectively to adult new neurons5,6, using a retroviral vector, in mice oxed for a gene of interest. Floxed genes are inserted with target sequences of Cre recombinase, called the loxP sequences. Cre recombinase recognizes the loxP sequences and removes the gene of interest. Using this technique, we prevented the expression of NMDA-type glutamate receptor (NMDAR), which receives signals from synaptic inputs, only in a small number of new neurons3. The survival of new neurons was affected in the absence of NMDAR, whereas the survival of wild-type new neurons in the same animals was not. Thus, we demonstrated that NMDAR-mediated regulation of new neuron survival is input-dependent and cell-specic. Further, as shown in this same study, the single-cell knockout technique is advantageous not only to reveal the cell-specic regulation, but also to indicate a competitive mechanism in the survival regulation, which is difcult to address with conventional global manipulations.
The single-cell gene knockout technique was designed specically to examine the behavior of manipulated single cells in intact environment. Therefore, this technique is suited for single-cell analyses such as morphological and patch-clamp analysis. On the other hand, because only a small number of new neurons are manipulated and the majority of new neurons are intact, this technique is not adequate for whole-animal studies such as behavioral experiments. One limitation of this single-cell knockout technique is the requirement of oxed mice generated for a specic gene of interest. However, currently, many such animals exist and laboratories continue to produce conditional knockout mice using the Cre/ loxP system4. It is advantageous in that this single-cell knockout technique is able to use these oxed mice produced for different purposes with a small additional effort. An alternative method, which is possibly more generally applicable, is retrovirus-mediated RNA interference. This approach was used to knock down a Na+K+-2Cl transporter, NKCC1, in adult new neurons7. One disadvantage of this method is that the expression of a gene of interest cannot be suppressed completely, in contrast with the complete elimination of the gene of interest in the knockout approaches. The other problem of this RNA interference-based approach is the difculty in verifying that an observed effect is really owing to knockdown of a gene of interest. For this purpose, it is necessary to use multiple sequences targeting the same gene and/or to perform rescue experiments with a homologous gene that is not affected by the sequences. This requires constructing multiple viral vectors and performing experiments with them. On the other hand, for the single-cell knockout technique, this verication can be more easily achieved by injecting the same viral vector in wild-type mice as we performed in the previous study3. A possible compliment to the retroviruses expressing Cre in oxed animals is to use promoters specic for new neurons with viral vectors such as lentiviral or adenoviral vectors. In this case, the specicity of promoters must be extremely high. Because new neurons are a minority in the adult brain (estimated B3% in the dentate gyrus) and intermixed with a
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large number of pre-existing mature neurons, even a small degree of nonspecicity could affect a much larger number of mature neurons than new neurons (T. Sawai, A. Tashiro & F.H.G., unpublished observation). All of these methods require high titer virus preparation and precise delivery of virus into the dentate gyrus. To prepare concentrated retrovirus for single-cell knockout, one needs an efcient transfection protocol and plasmid DNAs that are required for virus assembly, including the recombinant viral vector that contains the transgene, a plasmid that expresses gag and pol, and a plasmid that encodes an envelope protein. The wild-type retrovirus genomes contain three genes, gag, pol and env, encoding the structural, enzymatic and envelope proteins required for virus replication, respectively. In the recombinant retroviral vector system, these genes were separated from the virus genome so that the amplied virus is replication incompetent8. This has served two purposes. First, it is safer. Second, the gene expression is restricted to virus-transduced cells at the time of infection and the progeny of these cells, which allows the tracing of transduced cells. VSV-G is the envelope glycoprotein of the rhabdovirus vesicular stomatitis virus, and has been used to replace the envelope proteins of other viruses to increase the host range. In addition, VSV-G-pseudotyped virus is more stable under highspeed centrifugation9. Typically, a recombinant retroviral vector contains viral long terminal repeats at ends, the viral packaging signal, the promoter and gene of interest, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (Fig. 1). Our vector is based on the pCL system by Naviaux et al.10. It uses the cytomegalovirus (CMV) promoter to drive the transcription of the modied viral genome, which is based on the Moloney murine leukemia virus (MoMLV). The compound CAG promoter that contains chicken actin promoter, CMV enhancer and a large
CAG-GFP/cre Pcmv LTR
CAG GFP/cre WPRE
Figure 1 | A schematic structure of the recombinant retroviral genome of CAG-GFP/cre. This vector is based on the MoMLV. The transcription of the viral genome is under the control of CMV promoter to allow high-titer virus preparation with HEK293T cells. The expression of GFP/cre is driven by the compound promoter CAG, which contains the minimum enhancer sequence of CMV, chicken actin promoter and a synthetic intron. LTR: long terminal repeat; C: viral packaging signal; WPRE: Woodchuck hepatitis virus posttranscriptional regulatory element.
synthetic intron was used to allow the ubiquitous expression of GFP-fused Cre recombinase (GFP/Cre)11 in transduced cells3,6. The WPRE element is not an essential element of the virus backbone, but it has been reported to stabilize the transcripts and increase the expression level of transgene by several folds12. Because all three plasmid DNA need to be present in the same cell for the assembly of the virus, the transfection efciency is critical for generating high-titer virus preparation. We typically use the HEK293T cells for virus preparation and have used two methods in our laboratory, lipofectamine 2000 and calcium phosphate precipitation. The protocol with lipofectamine 2000 (Invitrogen) is highly consistent, less labor-intensive, but more expensive. In comparison, our protocol for calcium phosphate precipitation has been less consistent. We would recommend a test transfection to determine the efciency of the reagents before starting a retrovirus preparation with the calcium phosphate precipitation protocol. This protocol can be used for other applications of retrovirus to manipulate new neurons in the adult brain. For example, one can overexpress mutated genes, such as constitutively active or dominant-negative mutants or conditionally express genes by using regulatable promoters, such as the tetracycline-transactivator system13. A ow diagram of the procedure is shown in Figure 2.
REAGENTS. 68-week-old oxed mice ! CAUTION All animal experiments are to be performed in accordance with relevant authorities guidelines and regulations. Plasmid DNA: pCMV-gp (a gift from the laboratory of Inder Verma at Salk Institute), pCMV-vsv-g and CAG-GFP/cre3. The plasmids are available from the corresponding author. Culture medium: Dulbeccos modied Eagles medium (high glucose, with glutamine) or Iscoves modied Dulbeccos medium (Gibco, no. 12440-053) supplemented with 10% (vol/vol) fetal bovine serum. Transfection reagent: lipofectamine 2000 (Invitrogen, 11668-019), Opti-MEM (Invitrogen, 31950-062 or 31985-070). Alternative transfection reagent: 2 HEBS, 280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4, 12 mM D-glucose, 50 mM HEPES (acid-free), pH 7.05, adjust with NaOH or HCl, lter sterilize (do not autoclave), 2 M CaCl2, lter sterilize (wet lter with a few drops of water to allow ltration). Dulbeccos PBS (Invitrogen, 14287-072). Anesthetics (10 mg ml1 ketamine and 1 mg ml1 xylazine in 0.9% NaCl) ! CAUTION Controlled substances (e.g., ketamine) must be locked up when not in use and a controlled substance usage log must be kept on them.
. 70% ethanol. Cotton tips. Puralube Vet Ointment (Pharmaderm, NDC 0462-0211-38). Tissue adhesive (3M Vetbond, 1469SB). Biosafety level-2 facility ! CAUTION For general regulations of Biosafety
level-2 facility, please see pages 2127 of Biosafety in Microbiological and Biomedical Laboratories (http://www.cdc.gov/od/ohs/pdfles/ 4th%20BMBL.pdf). 14 ml polypropylene tubes (Falcon 352059). Ultracentrifuge (Beckman Coulter). Rotors for ultracentrifuge: SW32 Ti or SW28, SW55 Ti (Beckman Coulter). Polyallomer tubes mm (Beckman Coulter, cat. no. 326823) or mm konical (cat. no. 358126). Polyallomer tubes mm (Beckman Coulter, cat. no. 326819). Small animal stereotaxic frame (Kopf). Microsyringes (Hamilton, cat. no. 87925). Needles for microsyringes, 33 gauge (Hamilton, cat. no. 7762-06). Electric drill (Dremel, model 395 T6). Drill bur, size no. 1 (Henry Shein, cat. no. 100-7176). Electric hair trimmer or small scissors EQUIPMENT
PROCEDURE Preparation of high-titer retrovirus 1| Viruses can be prepared by lipofection (option A) or calcium phosphate transfection (option B).
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! CAUTION Pseudotyped MoMLV-based retrovirus is capable of Virus preparation Transfecting three plasmids into HEK293T cells with lipofection or CaCl2 method infecting human cells through contact. Gloves and protective Concentrating by ultracentrifugation clothing are required for working with retroviruses. Extra caution should be taken to avoid spill and splash when handling retrovirus-containing material. Retroviruses are labile and easily decontaminated by ethanol, detergent or bleach. Titer measurement Infecting retrovirus into HEK293T cells Working area should be decontaminated with ethanol or bleach after any spill or after the completion of work. ! CAUTION Replication incompetent retroviruses are considered to be infectious material, and biosafety level-2 Storage in 80 C freezer laboratories are used for virus preparation and manipulation. Follow the guidelines of NIH/CDC (reference: http://www.cdc. gov/od/ohs/pdfles/4th%20BMBL.pdf) and your institution for biosafety level-2 laboratories. Stream-sterilize all solid Virus injection Stereotaxic injection into the neurogenic area of adult floxed mice waste before disposal. Although cells and media are not infectious until the transfection step, we perform the entire procedures of virus preparation in the biosafety level-2 laboratories to avoid problems associated with transferring Analysis materials, such as contamination. Different maturational stages can be examined by different survival time after injection (A) Retrovirus preparation with lipofectamine 2000 (i) Day 1: The day before transfection, plate cells Figure 2 | Flow diagram of the protocol. per 100 mm plate for a total of 12 plates. This roughly equals 1/3 or 1/4 of one 100 mm plate of conuent 293T cells. Use medium including antibiotics when maintaining the 293T cells, but use medium without antibiotics at this step. Use 10 ml medium for each plate. (ii) Transfection (day 2) TIMING B6 h: Plan to transfect cells in the morning because there is a 5-h waiting time to change media. Prepare eight tubes (14 ml polypropylene tubes); add 2.4 ml Opti-MEM to each tube. (iii) To four tubes, add 45 mg DNA mix (containing 22.5 mg CAG-GFP/cre, 15 mg pCMV-gp and 7.5 mg pCMV-vsv-g) and mix well by tabbing the tube. (iv) To the other four tubes, add 150 ml lipofectamine 2000. Mix by tabbing the tube. m CRITICAL STEP Incubate at room temperature (2025 1C) for no longer than 5 min. (v) Mix one tube of DNA (Step 1A(iii)) with one tube of lipofectamine 2000 (Step 1A(iv)). m CRITICAL STEP Transfer DNA into the lipofectamine tube, pipette up and down for 23 times, and mix well by tabbing the tube. Do this one by one, and you will have four tubes of DNAlipofectamine mix with approximately 4.8 ml in each tube. Each tube transfects three of 100 mm plates. (vi) Incubate at room temperature for 2530 min. (vii) Transfer 1.6 ml of the DNA/lipid solution from Step 1A(vi) onto each plate of 293T cells containing normal culture medium without antibiotics, dispense the mix dropwise and have them evenly distributed on the surface of the cells. At the end, gently rock the plate and place the plate back to the incubator. You can do this in set of three. (viii) Incubate for 5 h at 37 1C, 5% CO2, then replace supernatant with 10 ml fresh media. Media containing antibiotics can be used at this step. (ix) Concentrate virus (day 4): collect virus. Start early. A total of B6 h is needed to complete the whole procedure, but there are some waiting times. ! CAUTION Handle retrovirus material with caution and avoid spills. Use bleach to decontaminate hazardous liquids (10% nal concentration for 30 min). All plasticware are to be stream-sterilized before disposal. (x) Collect the supernatant in 50 ml conical tubes. Centrifuge at 1,000g for 23 min to remove cell debris. (xi) Filter the supernatant through 0.22 mm lter top. It is not necessary to pre-wet the lters. (xii) Transfer ltered supernatant into four ultracentrifuge polyallomer tubes (mm, tubes A). Add PBS to the tubes so that the top of the solution is B0.5 cm from the top of the tube. (xiii) Centrifuge at 65,000g and 4 1C using the rotor SW32 Ti or SW28 for 2 h. (xiv) Remove the supernatant with Pasteur pipette. ! CAUTION Connect the ask to the venting system through a lter (Vacushield Vent Device, Pall Corp. no. 4402). (xv) Resuspend virus with 0.7 ml PBS for each tube (B2.8 ml total). Pipette up and down for 2030 timesadjust pipetteman to 0.5 ml to minimize air bubbles from pipetting. Transfer all to one 4 ml ultracentrifuge tube (mm, tube B). Sequentially wash tubes A with 0.7 ml PBS and transfer to tube B (3.5 ml now in tube B). (xvi) (Optional) Add 0.5 ml 20% sucrose cushion (made in PBS and lter-sterilized) to tube B.
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(xvii) Fill the tube with more PBS so that the top of solution is B0.5 cm from the top of the tube. Spin at 65,000g using the rotor SW55 Ti, 4 1C for 2 h. (xviii) Resuspend the nal pellet in 80 ml PBS by vortexing for 30 s and then by pipetting. This procedure is enough to resuspend the virus, although white or brown sticky pellet sometimes remains after resuspension. Transfer the virus to a 0.5 ml tube (tube C); wash tube B once with 20 ml PBS and transfer the solution to tube C. Spin tube C briey and transfer the supernatant to a new tube. Aliquot in 510 ml and store at 80 1C. ! CAUTION It is important that the virus solution is stored in small aliquots and stored at 80 1C. Virus solution can be at 80 1C for at least 1 year without signicant change in virus titer. Do not re-freeze virus solution after thawing. The virus solution can be stored at 4 1C if an experiment is planned within a week. (B) Retrovirus preparation by calcium phosphate precipitation (i) Again, the transfection efciency with calcium phosphate precipitation has not been consistent in our hands. It is better to conduct a test transfection before starting the preparation. Ideally, a transfection efciency of 450% is needed to have a good titer. On day 1, plate 293T cells at per 150 mm plate. Prepare 1020 plates for virus preparation. Use 15 ml media for each plate. Incubate cells with 10% CO2. Calcium phosphate tranfection (day 2, late afternoon, 46 pm) (ii) For each of the ve plates, add to a 50 ml conical tube (3.2-X) ml sterile ddH2O, where X is the volume of your DNA mix including 150 mg CAG-GFP/cre, 100 mg pCMV-gp and 4050 mg pCMV-vsv-g. (iii) Add DNA to the water and mix by tabbing the tube. (iv) Add 1.8 ml CaCl2 solution to the tube, mix by pipetting up and down three times. Tab on the tube for a few times. The total volume of DNA/CaCl2 mix is 5 ml. (v) Add 5 ml 2 HEBS solution to the DNA/CaCl2. m CRITICAL STEP Mixadd from high above (for example, position the tip of the pipette a little above the opening of the conical tube). (vi) Mix by pipetting up and down three times and then by bubbling air for 30 s. (vii) Incubate the mixture for 10 min at room temperature. (viii) Add 2 ml of the mix to each plate. m CRITICAL STEP Add dropwise and evenly on the plate. (ix) Distribute evenly by gently rocking the plate. (x) Incubate at 37 1C in 5% CO2 overnight. (xi) Day 3 early morning (810 a.m.): change media, and move cells back to 10% CO2 incubator. (xii) Day 4: collect the supernatant with 50 ml conical tubes. Add fresh media to plates and put cells back to 10% CO2. ! CAUTION Use bleach to decontaminate hazardous liquids. All plasticware are to be stream-sterilized before disposal. (xiii) Day 5: collect the supernatant again and discard cells. (xiv) Concentrate virus following Steps 1A(ix)(xvii). Two ultracentrifuges might be needed because of the large volume of total supernatant. For example, a total of 300 ml supernatant will be collected if ten of 150 mm plates are used for virus preparation. Each rotor can only accommodate 180200 ml. Determine the titer of the virus 2| Seed 105 293T cells per well in 24-well plate 1 day ahead. This is about 1/200 of the amount of cells from one conuent 10 cm dish. Seed four wells for one virus preparation. ! CAUTION Vsvg-pseudotyped MoMLV-based retrovirus is capable of infecting human cells through contact. Gloves and protective clothing are required for working with retroviruses. Extra caution should be taken to avoid spill and splash when handling retrovirus-containing material. Retroviruses are labile and easily decontaminated by ethanol, detergent or bleach. Working area should be decontaminated with ethanol or bleach after any spill or after the completion of work. 3| Steps 3 and 4 are carried out in the virus laboratory. Perform a serial dilution (102, 103, 104 and 105) of the virus and transfer 10 ml of each dilution to one well so that the nal dilution series are 101, 102, 103 and 104. 4| Count the number (n) of uorescent clusters under the uorescent microscope 23 days after viral infection. The virus titer is (ndilution/10) colony-forming unit (c.f.u.) per ml. Typically, one could obtain a titer of 104105 c.f.u. ml1, that is 107108 c.f.u. ml1. p24 measurement by real-time PCR or ELISA can be an additional or alternative option to measure the titer of the retrovirus14. Injection of virus vectors into the dentate gyrus 5| Inject anesthetics into 68-week-old oxed mice of interest. Wait for 5 min so that the mice do not respond to pinching with tweezers. ! CAUTION All animal experiments are to be performed in accordance with relevant authorities guidelines and regulations.
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Figure 3 | Stereotaxic injection. (a) After a mouse is mounted on a stereotaxic frame and its skull is exposed, the bregma is localized (arrowhead). Antero-posterior (AP) axis is indicated on left. (b) The needle tip is moved onto the bregma (arrowhead). (c) The tip is moved posteriorly and laterally to an appropriate stereotaxic coordinate. The position of the tip (arrow) is marked by a marker pen. (d) A small hole (arrow) is made in the skull by using an electric drill. (e) The needle is brought down to an appropriate coordinate through the hole (arrow).
! CAUTION Retrovirus is infectious to human by contact. Wear Latex gloves when handling the virus. Avoid spills. Thoroughly decontaminate equipment with 70% alcohol after use. 6| Shave a small area on the head with a trimmer. Apply eye ointment (Pura lube Vet Ointment) to prevent eyes from over-drying. 7| Cut the skin over the skull about 1 cm. Wipe blood from the wound. 8| Mount the mouse onto a stereotaxic frame. m CRITICAL STEP Orienting the head straight in terms of the antero-posterior axis and horizontally is important for consistent injection into the target area. 9| Move the tip of injection needle to the bregma. See Figure 3. 10| Move the needle tip to 1.5 mm lateral and 2 mm posterior. Mark the position with a marker pen. The stereotaxic coordinate shown here (Steps 10 and 13) is for 68-week-old oxed NR1 mice and C57Bl/6 mice used by Tashiro et al.3. The coordinates need to be adjusted according to the line and age of mice used for each study. 11| Move the needle out and make a small hole on the skull using an electric drill. 12| Dip the needle tip into virus solution and load 1.5 ml of solution into the syringe. 13| Move the needle tip to the hole and set it to the level of skull surface. m CRITICAL STEP Move the needle tip down by 2.3 mm. 14| Inject a total of 1.5 ml of virus solution. m CRITICAL STEP We manually inject 0.05 ml every 6 s. 15| After nishing the injection, wait for 1 min to prevent the injected solution from owing back through the needle track. 16| Move the needle tip 1 mm up and then wait for another 1 min. 17| Move the needle tip 1 mm up and the tip is out of the brain. Rinse the needle with 70% ethanol and PBS sequentially. 18| Close the skin using tissue glue or by sewing with thread. Place the mouse in a cage on top of a warm blanket until it is fully alert. Transfer the mouse back to home cage. 19| Analysis. A variety of single-cell analysis is possible. For example, neuronal differentiation and morphogenesis can be
Figure 4 | New neurons expressing GFP/cre in the adult dentate gyrus. (a) A new neuron in adult ROSA26 GFP reporter mice. Because GFP/cre is localized in the nucleus, GFP uorescence (green) in the cytoplasm, most evidently in dendrites, indicates the occurrence of Cre-mediated recombination in the new neuron. Red: anti-NeuN immunostaining. (b) A new neuron in adult oxed NR1 mice, cotransduced by two viral vectors (CAG-GFP/cre and Red uorescent protein (RFP)). A subpopulation of new neurons coexpressed GFP/cre (green) and RFP (red). Blue: anti-Prox1 immunostaining. Scale bar, 30 mm in (a), 60 mm in (b). NATURE PROTOCOLS | VOL.1 NO.6 | 2006 | 3053
analyzed in xed brain sections with immunostaining3,5,6. Electrophysiology and morphological dynamics can be studied in live brain slices3,5. Importantly, depending on survival time after virus injection, new neurons at different maturational stages can be analyzed3,6. We have analyzed from 1 day to 14 months after virus injections. TIMING Steps 1A(i)(xviii): virus preparation, 4 days (30 min for day 1, 6 h for day 2 and 6 h for day 4) Steps 1B(i)(xiv): virus preparation, 5 days (3060 min for day 1, 60 min for day 2, 30 min for day 3, 30 min for day 4 and 68 h for day 5) Steps 24: 4 days (15 min for day 1, 30 min for day 2 and 1020 min for day 4) Steps 518: virus injection, 15 min to 1 h for each mouse (depending on the experimenters experience)
? TROUBLESHOOTING Troubleshooting advice can be found in Table 1.
TABLE 1 | Troubleshooting table. Problem Step 1A(xiii)tubes deform after spinning Possible reason Adapters are not used with conical polyallomer tubes Tubes are not lled with solution Solution Use adapters Fill the tubes with PBS so that the surface of the solution is B0.5 cm away from the tube opening Make several 2 HBS solutions and test the efciency of each of them. Use the one that gives the maximum transfection efciency (at least 50%) Prepare a new batch of virus with a high titer Correctly mount mice on the stereotaxic frame Find a good stereotaxic coordinate by injecting a dye (e.g., gel-loading dye for electrophoresis) into the same line of mice at the same age as used for specic studies. The size of brain is different between lines of mice and different ages
Step 1Blow transfection efciency
pH of 2 HBS might not be optimal
No GFP/cre-positive cells found GFP/cre-positive cells only outside of targeted area
The titer of the virus is too low Injection into wrong area
ANTICIPATED RESULTS CAG-GFP/cre was injected into ROSA26 GFP reporter mice and oxed NR1 mice (Fig. 4). In ROSA26 GFP reporter mice, we found that Cre-mediated recombination occurred in more than 97% of GFP/cre-expressing neurons. In fNR1 mice, none of the neurons examined responded to an NMDA receptor agonist. Thus, in these mice, recombination efciency was quite high. The density of GFP/cre-expressing new neurons is higher at a position closer to injection sites, and the average density typically ranged from 4 to 10 cells per 40-mm-thick coronal sections with an injection of 1.5 ml virus (107108 c.f.u. ml1). As controls, one can use CAG-GFP in separate mice or CAG-RFP in the same mice. With the latter, an issue of variability from animal to animal can be reduced.
COMPETING INTERESTS STATEMENT The authors declare that they have no competing nancial interests. Published online at http://www.natureprotocols.com Reprints and permissions information is available online at http://npg.nature.com/ reprintsandpermissions 1. Gage, F.H. Mammalian neural stem cells. Science 287, 14331438 (2000). 2. Ming, G.L. & Song, H. Adult neurogenesis in the mammalian central nervous system. Annu. Rev. Neurosci. 28, 223250 (2005). 3. Tashiro, A., Sandler, V.M., Toni, N., Zhao, C. & Gage, F.H. NMDA-receptormediated, cell-specic integration of new neurons in adult dentate gyrus. Nature 442, 929933 (2006). 4. Tsien, J.Z. et al. Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87, 13171326 (1996).
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