Targeted Knockdown of Genes in the Choroid Plexus

The objective of this research is to investigate the techniques for manipulating gene expression in the choroid plexus. Our study has introduced two methods that allow for technique exclusively in the choroid plexus without any discernible impact on other regions. Our findings can potentially enhance future function studies of the choroid plexus in neuroscience.

Our research has reviewed that inhibiting T-cell infiltration across the choroid plexus through selective lockdown of adenosine to a receptor is sufficient to protect mice from EAE pathology. This finding supports the notion that detecting gateway activity in the choroid plexus could present a promising new strategy for treating multiple sclerosis. This study presents two alternative methods for gene expression manipulation in the choroid plexus.

ICV injection of Cre recombinase into those transgenic mice or ICV injection of AAV2/5, or it’s targeted SHI in the WT mice. These methods overcome issues with promoting specificity and transgenetic mouse breeding. To begin, place the anesthetized mice on the surgical platform.

After trimming the hair, apply eye ointment down both eyes to prevent them from drying. Then fix the mice onto a stereotaxic apparatus for immobilization. Make a small incision under a microscope to fully expose the skull.

Prepare two 10 microliter syringes, one containing AAV2/5 adenosine 2A receptor shRNA, and the other containing AAV2/5 scramble. Then use the microscope to locate the bregma and lambda, and set the coordinate point on the 10 microliter syringe based on the mouse brain stereotaxic atlas. Drill a small hole in the skull at the established coordinate point.

Inject two microliters of AAV2/5 adenosine 2A receptor shRNA virus into the brain ventricle via lateral injection at a steady rate of 100 nanoliters per minute. Keep the needle in place for 10 minutes before withdrawing. Seal the injured skin promptly using medical bio fibrin glue to prevent virus loss.

Place the mouse on a heating pad, maintaining its body temperature approximately at 37 degrees Celsius to facilitate recovery. Inject two microliters of CRE-TAT Recombinase into each lateral ventricle of a Rosa-LDL-td Tomato mouse for the experimental group, and two microliters of sterile PBS for the control group. Two weeks after the CRE-TAT recombinase injection, prepare frozen tissue sections, perform nuclear staining, and prepare the slide for imaging.

Knocking down adenosine 2A receptor in the choroid plexus in homozygous adenosine 2A receptor floxed mouse using CRE-TAT led to decreased EAE clinical scores. Similarly, knocking down choroid plexus and adenosine 2A receptor wild-type mice using AAV2/5-shRNA decreased EAE clinical scores. The QPCR analysis showed that the mRNA levels of adenosine 2A receptor decreased in the AAV2/5-shRNA and CRE-TAT groups compared to each control.

Silencing adenosine 2A receptor specifically in the choroid plexus reduced immune cell infiltration into the spinal cord.