A Phenotyping Regimen for Genetically Modified Mice Used to Study Genes Implicated in Human Diseases of Aging

The overall goal of this testing regimen is to establish a series of simple tests that can be used as an initial phenotyping pipeline to direct subsequent detailed investigations into behavioral phenotypes and the structure and function of tissues. Whether the behavioral and cellular phenotypes of human diseases are recapitulated in the mouse model. The main advantage of this technique is that the tests can be performed in laboratories with no prior experience in such experiments without significant investment yet still informed further detail studies.

Conduct the Hind Limb Test daily from post-natal day four to 10 to evaluate proximal hind limb muscle strength, weakness and fatigue. To begin, let the mice acclimate to the test area for 30 minutes. Meanwhile prepare a new 500 milliliter conical tube by washing it out with 70%ethanol and then pushing one or two cotton balls into the bottom of the tube.

Secure this tube in a vertical position for the test. When not being tested, keep the pups with the dam to maintain their temperature. After the half-hour acclimation, remove a pup from the cage and record its weight.

Then gently suspend the neonate by its hind limbs over the rim of the tube, so the pup is facing the cotton. Then count the number of pull attempts made by the pup and measure its latency to fall. After waiting 30 seconds, repeat the test.

After the second test, label the pup with a marker. Then rub the pup gently with bedding from the home cage and return it to the dam. Because older P8 pups can not be marked identify them with a toe clipping.

Before proceeding to the next pup, wipe the test tube down with 70%ethanol. Administer the Weanling Observation Test daily from P19 to P21 to score movement and general activity levels. Begin with letting the mice acclimate to the test area for 30 minutes.

While the pups acclimate, prepare the arena. Mark a plexiglass testing box with a six-by-six grid of two inch squares. Before using the box, clean it with 70%ethanol.

Then position a video camera so a side view of the whole box fills the field of view. Standardize the appearance of the surroundings to avoid introducing novelty. Before the test, transfer weanling to a clean holding cup and record its weight.

Then began video recording. First, film a test identification card with the mouse identification number, its age and the current date. Then transfer the mouse from the holding cup to the center square.

For three minutes, count the number of lines crossed by both front paws. After three minutes, return the mouse to a clean cage and end the video recording. Between every test, clean the box with 70%ethanol.

From the videos, count the rearing events, which is when a mouse stands with both front paws clear of support. Also count the grooming events, which occur when both front paws are engaged in cleaning. Conduct the Grip Strength Test on alternate days between P22 and P26 to assess neuromuscular strength.

As usual, give the mice 30 minutes to adapt to the test area. The testing apparatus is a plexiglass box with a mesh grid. Clean both with 70%ethanol.

Then line the bottom of the box with a layer of bubble wrap for padding and cover the bubble wrap with paper to absorb excretions. Proceed to separate the test mice and load them into individual cups. Now, place the first mouse onto the center of the wire mesh and slowly invert the mesh to 10 to 20 centimeters above the soft surface of the box.

Measure the latency to falling or end the test after 60 seconds, if the mouse does not fall. Then return the first mouse to the holding cup. Between tests, wipe the mesh with 70%ethanol and replace the paper over the bubble wrap.

Then conduct the test on the two other mice in the group. After each mouse is tested once, repeat the process twice more for three trials per mouse. If there are fewer than three mice in a group, make sure that there is an inter-trial recovery time of at least five minutes.

For an NADH Diaphorase Stain combine equal volumes of freshly made NADH solution and freshly made NBT solution. Then add one milliliter of staining solution to each slide and incubate the slides in a humidified chamber at 37 degrees Celsius for 30 minutes. For a Succinate Hydrogenase Stain, prepare a solution of 0.1 molar sodium succinate, 1.25 molar NBT and 0.2 molar phosphate buffer.

Then add one milliliter to each slide. Pack the slides into a humidified chamber and incubate the slides at 37 degrees Celsius for an hour. After incubating with either stain, aspirate the staining solution and wash the slides three times with distilled water.

Then wash the slides through a series of acetone baths that increase and decrease in concentration. With each concentration of acetone, perform three washes for two minutes per wash. Lastly, rinse the slides three times in distilled water.

Then mount the slides with an aqueous medium. Mice without HTRA2 expression were examined using the described battery of tests and compared with litter mate controls. No change in the number of pulls or the latency to fall was seen between P4 and P6.Between P7 and P10, a reduction in neuromuscular strength was noted.

In the activity test, there was reduced activity of HTRA2 knockout mice. HTRA2 knockout mice also showed a reduction in strength in the wire mesh grip test. H and E staining show that the HTRA2 knockout mice had no apparent changes in retinal structure compared to controls at P35.

Finally, histochemical staining for electron transport chain complex activity was used to identify changes in respiratory enzyme functions. There was decreased staining in the HTRA2 knockout mice compared to controls. This was noted in the cerebellum and striatum, but not the cortex.

After watching this video, you should have a good understanding of how to assess the activity and neuromuscular strength of genetically modified mice, as well as how to combine these studies with cellular axes. Thanks for watching and good luck with your experiments.