Establishing this humanized mouse model can aid in preclinical HIV research studies. For example, both different viral strains and novel HIV interventions can be evaluated. These protocols can be used to evaluate stem cell donor CCR5 variant status and to efficiently infect humanized mice with HIV and to quantify mouse plasma viral loads.
The RNA quantification protocol can be easily adapted to quantify any viral RNA sequence of choice that is detectable in plasma samples. For genetic screening of cord blood samples for CCR5 delta 32 variants, incubate 1.25 microliters of non-pelleted flowthrough with 11.25 microliters of PCR mix containing 200 micromolar dNTP mix, 0.01 unit per microliter of high-fidelity DNA polymerase, and forward and reverse primers as detailed in the table. Adjust the volume with nuclease-free water to approximately 12.5 microliters for each PCR reaction and amplify the genomic fragments with the PCR cycling program as indicated in the table.
At the end of the reaction, separate the PCR products on a 2%agarose gel. PCR products from the wild type alleles and the delta 32 alleles yield PCR fragments of 196 base pairs and 164 base pairs respectively, making the bands easily distinguishable by gel electrophoresis. For intravaginal HIV exposure, place a heating lamp focused on the center of the workspace where the mouse will be located and place sterile 20 microliter pipette tips in an appropriate pipette into the hood.
Place a container with liquid disinfectant into the hood for immediate inactivation of any materials and liquids that have been in contact with the virus. Place the anesthetized mouse onto a sterile blue pad in the supine position under the lamp with the tail of the mouse facing the back of the hood and confirm the lack of response to pedal reflex in the mouse. Grasp the animal at the base of the tail.
Gently lifting the mouse by the tail base, support the animal in an upside down upright position. Use a sterile pipe tip to stimulate the genital area with gentle stroking up toward the anus to induce emptying of the rectum and to relieve pressure on the vagina. To expose the vaginal opening, carefully wrap the mouse tail across the fingers, such that the vulva naturally opens.
Using a new pipette tip, place the tip at the level of the vaginal opening and atraumatically release 20 microliters of the virus into the vagina, allowing gravity to pull the viral suspension into the vagina. When all the virus has been delivered, retain the mouse in this position for five minutes to avoid gravity-induced leakage of the virus. Then, carefully return the mouse to its home cage in the supine position and monitor until full recovery.
To isolate RNA from thawed mouse plasma samples, use a virus RNA isolation kit according to the manufacturer's instructions. Add the sample that binds to the column and add an on-column DNase treatment step to ensure the removal of all DNA within the plasma sample. Then store the RNA samples at 80 degrees Celsius for at least one hour.
To synthesize cDNA, use reverse transcriptase according to standard protocols, including 0.5 microliters of an RNase inhibitor within the cDNA reaction to avoid RNA degradation, then perform cDNA synthesis as detailed in the table and store the cDNA samples at 80 degrees Celsius for at least one hour. To prepare samples for ddPCR, mix 11 microliters of ddPCR probe reaction mixture containing polymerase and dNTPs with 250 nanomolar of minor groove binding probe and 900 nanomolar of each of the forward and reverse primers, as indicated in the table. Add three microliters of each cDNA sample and adjust the total PCR volumes to 22 microliters with nuclease-free water.
Use droplet generation oil for probes on a droplet generator according to the manufacturer's protocol to emulsify the PCR mixes. Then run the PCR program as outlined in the table. For cART treatment after confirmed HIV infection, feed the mice with pellets prepared by an external vendor containing a standard cART regimen, as indicated in the table.
Use a red-colored cART diet to easily distinguish it from ordinary mouse chow and use a control chow diet without cART in a standard brown color. For initiation of the treatment, place the cART-containing chow diet into sterile cages before transferring the mice from the old cage to the new cage. Then monitor the weights of the mice and check the consumption of cART-containing chow daily to ensure that the mice are adjusting to the change.
Here, the flow cytometry gating strategy for analysis of the stem cell purity is depicted, with the purity of the isolated CD34 positive stem cells typically ranging between 85 to 95%with a less than 1%T cell contamination. PCR analysis demonstrates CCR5 variant status of the purified donor stem cells, making CCR5 delta 32 heterozygous donors easily identifiable. The frequency of the mutant genotype in a group of 19 donors was around 15.8%in agreement with larger epidemiological studies of Scandinavian populations.
Three to five months after the adoptive transfer of 7.5 times 10 to the 4th human CD45 positive cells into recipient mice, 20 to 50%of the white blood cells recovered from recipient animal peripheral blood samples are human CD45 positive cells and the mice will have developed human B and T cells. 64%of mice become infected with HIV following vaginal exposure as demonstrated. Four weeks after switching to a 40 day diet containing standard cART, the viral loads in HIV positive mice decrease below the detection limit.
After cessation of the diet, the virus rebounds, mirroring clinical data. Importantly, mice on cART tolerate the change in diet well. It is crucial to remember that these animals are immunodeficient and therefore vulnerable to the environment.
Adhering to aseptic techniques will increase the overall animal health and protocol success. After establishment of the humanized mouse cohort for HIV infection, the protocol may be adapted for viral RNA quantification in tissues or for testing novel interventions in different viral strains. The flexibility and accessibility of humanized mice for HIV infection studies have enabled critical progress in HIV virology, immunology, and treatment research.
Before attempting protocols involving infectious HIV, be sure to obtain approval from local work environment officials and to adhere to approved decontamination procedures.
