Cloning Plasmids for Gene Editing in Human Lung Cells
Session Number
Project ID: BIO 12
Advisor(s)
Preetish Kadur Lakshminarasimha Murthy, University of Illinois at Chicago
Discipline
Biology
Start Date
17-4-2024 8:35 AM
End Date
17-4-2024 8:50 AM
Abstract
The purpose of gene editing is to manipulate the expression levels of a gene of interest. The focus of this experiment was to manipulate TP63 expression levels between proximal and distal epithelial airway cells. Gene cloning was performed through restriction enzyme digest cloning, in which the non-region of interest is cut off and substituted with the gene sequence of interest selected by software. Three different cloning techniques were used: CRISPR Cas9, Open Reading Frame, and short hairpin RNA. From each cloning technique two sets of plasmids were made, one for control and one to change gene expression levels. A virus was made using each of the different gene editing techniques and introduced to human epithelial cells. Transduced cells were sorted and had their RNA extracted to determine if gene cell expression levels were changed after virus introduction. Based on preliminary data, we can transduce primary human cells with high efficiency using the Open Reading Frame method and have seen a change in gene expression, but further replicates have to be performed to show significance. Future steps include infecting more than three samples and exploring how the cell integrity has changed by differentiating infected cells into different cell types.
Cloning Plasmids for Gene Editing in Human Lung Cells
The purpose of gene editing is to manipulate the expression levels of a gene of interest. The focus of this experiment was to manipulate TP63 expression levels between proximal and distal epithelial airway cells. Gene cloning was performed through restriction enzyme digest cloning, in which the non-region of interest is cut off and substituted with the gene sequence of interest selected by software. Three different cloning techniques were used: CRISPR Cas9, Open Reading Frame, and short hairpin RNA. From each cloning technique two sets of plasmids were made, one for control and one to change gene expression levels. A virus was made using each of the different gene editing techniques and introduced to human epithelial cells. Transduced cells were sorted and had their RNA extracted to determine if gene cell expression levels were changed after virus introduction. Based on preliminary data, we can transduce primary human cells with high efficiency using the Open Reading Frame method and have seen a change in gene expression, but further replicates have to be performed to show significance. Future steps include infecting more than three samples and exploring how the cell integrity has changed by differentiating infected cells into different cell types.