| 制作方法Making method |
NOD-Rag1tm1Mom Il2rgtm1Wjl (NRG) mice was obtained from Jackson Laboratories.Umbilical cord blood was obtained with parental consent from healthy newborns in the Department of Obstetrics and Gynecology at the McMaster University Medical Centre. Te umbilical cord blood was processed using the RosetteSep Human Cord Blood Progenitor Enrichment Kit from Stem Cell Technologies (Vancouver, BC, Canada), to purify and enrich CD34+ cells. Purifed CD34+ cells were cryopreserved until required. Four day old NRG pups were sub-lethally irradiated with a single-dose γ-ray (300 cGy) and then administered an intrahepatic injection with 1×10^6 to 2×10^6 CD34-enriched hematopoietic stem cells. Te degree of human immune cell reconstitution in the NRG mice was evaluated by fow cytometry 90–120 days following the intrahepatic injection by quantifying the expression of human leukocyte markers, hCD45, hCD3, hCD4, as well as mouse CD45 in peripheral blood mononuclear cells (PBMCs) to determine the percent reconstitution in each Hu-mouse. Hu-mice in the progesterone high/diestrus stage of the estrous cycle were anesthetized by IP injection of anesthetic (150 mg ketamine per kg/10 mg xylazine per mL). Mice were placed in a supine position, the vaginal tract was gently swabbed and an IVAG inoculation with a 25 µl dose of 10^3 (low) to 10^5(high) TCID50/mL of NL4.3-Bal-Env HIV-1 was delivered, by gently inserting the viral inoculum using a pipette. |
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