Rapid Genotyping of Animals Followed by Establishing Primary Cultures of Brain Neurons

The overall goal of this procedure is to genotype newborn mice and to generate low density neuronal cultures on a glial feeder layer. This is accomplished by first rapidly genotyping newborn mice using their tail tips in the second step. On the same day, the brains are dissected and cultured as glial feeder layers.

Then after a few weeks, another group of newborn mice are rapidly genotyped and neuronal cells are generated from the animal's brains for culture on the preceded glial feeder layers. Ultimately, this method can be used for rapid and reliable genotyping as well as for the generation of healthy neurons and glial cells. There are two main advantages of this protocol.

One is that we can culture healthy neurons at a very low density for imaging experiments by plating them on a glial fetal layer. The second advantage is that newborn mice can be genotyped in several hours, allowing matching of the genotypes of the neurons and the glial fetal layer. I'm ko a research assistant in the Harta lab.

I'll demonstrate the procedures for genotyping and cultures. Begin by adding 200 microliters of DNA extraction solution to each PCR tube containing a specimen. Place the tube strip into a PCR thermal cycler and then start the DNA extraction when the extraction is finished.

Remove the tube strip from the thermal cycler and invert the tubes five times. Then transfer four microliters of DNA extract from each specimen to an unused tube on an eight tube strip and mix the sample with 10 microliters of two XPCR ready. Mix two, two microliters of mixed forward and reverse primers, and four microliters of nuclease free water.

For each specimen, place the samples into a thermal cycler and start the thermal cycling program. When the program is finished, load 20 microliters of each amplified sample or molecular weight marker directly into the appropriate well of an aros gel. Then run the gel and acquire fluorescent images of the bands under ultraviolet light.

To detect the amplified DNA products, begin by removing the regions of interest from each hemisphere of the brain and then transfer the brain tissues into a 15 milliliter tube. Let the tissue settle at the bottom of the tube and then replace the solution from the top of the slices with five to 10 milliliters of ice cold. Hank solution with or without fetal bovine serum as indicated before, completely aspirating all of the solution.

After the fourth wash, use a three milliliter syringe to strain two milliliters of freshly prepared tryin containing digestion solution through a 0.2 micrometer syringe. Filter directly into the tube containing the brain tissues and let the trypsin ization proceed at room temperature. After 13 minutes, neutralize the reaction by first aspirating most of the trypsin solution and then adding seven to 10 milliliters of ice cold hank solution with fetal bovine serum.

Then rinse the tissues in Hank solution with or without fetal bovine serum as just demonstrated. Next, filter two milliliters of dissociation solution onto the brain tissues. Then mechanically dissociate the cells with gentle trier 10 to 20 times using a cotton plugged fire polished PEs your pipette.

Continue the trier until the visible tissue pieces disappear. Wait three minutes for the small pieces to settle down and then use the PEs pipette to transfer about 1.5 milliliters of the solution into a 15 milliliter conical tube containing three milliliters of ice cold Hank solution with fetal bovine serum spin down the tubes for 13 minutes at 185 Gs and four degrees Celsius. Then carefully aspirate the supernatant using the same pasteur pipette.

Gently resuspend the pellet in one milliliter of prewarm plating medium one. Then add approximately four milliliters of prewarm plating medium. Transfer the cell suspension to an uncoated T 25 culture flask and place the flask in a cell culture incubator.

At one day in vitro, rinse the cells twice with five milliliters of ice cold plating medium, one by gently tilting the flask several times in a swirling motion at six to nine days in vitro place 100 microliters of coating material with extracellular matrix proteins onto glass cover slips in a cell culture dish and place the dish in the cell culture incubator. The next day, rinse the flask by aspirating all the solution within the flask and adding approximately 13 milliliters of ice cold hank solution. Then aspirate the solution completely.

Next, filter four milliliters of a trypsin EDTA plus DNA solution directly onto the cells and allow the cells to dissociate from the cell culture flask in a 37 degree Celsius incubator. After 13 minutes, neutralize the reaction with two milliliters of ice cold, 100%fetal bovine serum, and use a five to 10 milliliter pipette to transfer the cell suspension into a 15 milliliter conical tube. Then wash the cells by adding approximately four milliliters of ice cold hank solution with fetal bovine serum centrifuging, and then resus suspending the pellet in one milliliter of prewarm plating medium.One.

Now aspirate the coating material completely from the cover, slips and plate approximately 50 microliters of the resuspended glial cells onto the cover slips. To establish the glial feeder layer, incubate the glial cells for 20 to 60 minutes at 37 degrees Celsius, and then add one milliliter of prewarm plating medium, one to each cover slip while the plating medium is added. It is useful to use another pipette to press down on the periphery of the cover slip so that the cover slip will not float in the medium.

After overnight culture. Replace the medium with one milliliter of fresh prewarm plating medium, one at two to three days. In vitro, add 10 microliters of mitotic inhibitor to each feeder layer culture to inhibit DNA replication and glial cell proliferation at seven to nine days.

In vitro, replace the culture medium with prewarm plating medium two. Then gently plate approximately 50 to 100 microliters of mouse neuronal cells in prewarm plating medium two onto each glial feeder layer. In this image, the tour one a gene was amplified from genomic DNA, isolated from the tail clips of newborn, of wild type heterozygous and homozygous pups of the knockin DYT one dystonia model.

Separate bands for the wild type and mutated alleles are clearly visible. In this figure two representative fields from a mouse hippocampal neuronal culture are shown. They were generated from the CA three, CA one hippocampal region of wild type mice.

14 days after plating on a pre-established ca three, ca one hippocampal glial feeder layer of wild type mice in the differential interference contrast images, signs of good health, such as a clear margin of neuronal cell body extended neuronal processes, and a relatively uniform layer of glial cells can be observed. Further immuno cyto chemical staining for the neuronal marker map. Two, identifying the neurons and their processes in each field can be observed as well.

Using this protocol, a genotyping in one round of culture can be completed in 67 hours. This protocol is especially useful for culturing neurons of genetically engineered mice that can die several days after birth.