Knocking Out the β-catenin from Induced Astrocytes using CRISPR/Cas9 Lentiviral Approach
Session Number
Project ID: MEDH 30
Advisor(s)
Dr. Srinivas D. Narasipura; Rush University
Dr. Tanner Shull; Rush University
Dr. Lena Al-Harthi; Rush University
Discipline
Medical and Health Sciences
Start Date
19-4-2023 11:25 AM
End Date
19-4-2023 11:50 AM
Abstract
Astrocytes are one of the most abundant cell types in the central nervous system (CNS), playing an important role in regulating glutamate uptake, neuroinflammatory response, blood brain barrier permeability, and neuronal health. In the past decade, hiPSCs (human induced pluripotent stem cells) were successfully demonstrated to differentiate into induced astrocytes (iAs), and recently have been shown to robustly express the Wnt/β-catenin pathway. The pathway is an important pro-survival that has been seen to regulate vital functions in primary astrocytes, such as glutamate uptake, immunoinflammatory response, and viral transcription. To further investigate the pathway’s role in astrocytes, we attempt to knock out β- catenin from the astrocytes. CRISPR/Cas9 knockout was previously attempted via plasmid transfection but was unsuccessful. Here, we attempted CRISPR/Cas9 knockout via a lentiviral approach. Lentiviruses were produced using third generation packaging systems and from 293 T-cells. Knockout cells were selected with puromycin. Two guide RNAs were tested in addition to control, scramble guide RNA. While western blot showed successful knockout with one of the guide RNAs, NGS only showed a success rate of ~1 successful knockout among ~30,000 cells in the correct spot. The protocol, which was adapted from a protocol to knock out genes in intestinal cells via lentiviruses, may have to be modified to yield more successful knockout efficacy.
Knocking Out the β-catenin from Induced Astrocytes using CRISPR/Cas9 Lentiviral Approach
Astrocytes are one of the most abundant cell types in the central nervous system (CNS), playing an important role in regulating glutamate uptake, neuroinflammatory response, blood brain barrier permeability, and neuronal health. In the past decade, hiPSCs (human induced pluripotent stem cells) were successfully demonstrated to differentiate into induced astrocytes (iAs), and recently have been shown to robustly express the Wnt/β-catenin pathway. The pathway is an important pro-survival that has been seen to regulate vital functions in primary astrocytes, such as glutamate uptake, immunoinflammatory response, and viral transcription. To further investigate the pathway’s role in astrocytes, we attempt to knock out β- catenin from the astrocytes. CRISPR/Cas9 knockout was previously attempted via plasmid transfection but was unsuccessful. Here, we attempted CRISPR/Cas9 knockout via a lentiviral approach. Lentiviruses were produced using third generation packaging systems and from 293 T-cells. Knockout cells were selected with puromycin. Two guide RNAs were tested in addition to control, scramble guide RNA. While western blot showed successful knockout with one of the guide RNAs, NGS only showed a success rate of ~1 successful knockout among ~30,000 cells in the correct spot. The protocol, which was adapted from a protocol to knock out genes in intestinal cells via lentiviruses, may have to be modified to yield more successful knockout efficacy.