S-Space College of Engineering/Engineering Practice School (공과대학/대학원) Dept. of Electrical and Computer Engineering (전기·정보공학부) Theses (Ph.D. / Sc.D._전기·정보공학부)
Localized viral gene delivery using encoded microparticles for high-throughput high-content cell-based assays
고속 다중변수 세포기반 분석을 위한 코드화된 미세입자를 이용한 지역화된 바이러스 기반의 유전자 전달
- 공과대학 전기·컴퓨터공학부
- Issue Date
- 서울대학교 대학원
- Cell-based assays; High-content screening; Multiplexing; Viral gene delivery; Encoded microparticle
- 학위논문 (박사)-- 서울대학교 대학원 : 전기·컴퓨터공학부, 2015. 8. 권성훈.
- In this dissertation, I develop an adenoviral vector-immobilised patch-type encoded microparticle for high-throughput, high-content cellular assays and name this encoded viral micropatch. This technology spatially confines the adenoviral gene delivery to only the cells under the micropatch by simply pipetting a heterogeneous mixture of the two-dimensional (2D) shape-coded viral micropatches on monolayer-cultured cells. Distinct clusters of transduced cells are then created in correspondence with the randomly positioned micropatches and the delivered gene into the cells within each cluster can be identified using the shape of the micropatch. For this purpose, shape-coded polymer microparticles are fabricated by photolithography, and highly localized gene delivery is achieved by specifically immobilizing adenoviral vectors on the microparticles. This unique feature allows high-throughput compound screening by virtue of multiplexing in a well of a standard microplate and creates no restriction for fluorescence-based assay formats with high-content imagers. To highlight the capabilities of this technology, I demonstrate a multiplex G-protein coupled receptor (GPCR) internalization assay that requires compound treatments followed by fluorescence-based target tracking at the sub-cellular level.
First, I develop the maskless lithography system supporting an automated step-and-repeat operation for the fabrication of microparticles with various 2D graphical codes. Using this system, I explore new applications of the encoded microparticles and lithography technique such as anti-counterfeiting of drugs, parallel loading of small volume liquid for multiplexed bioassays, and conformal phosphor coating for white light-emitting diodes (LEDs).
For the development of the encoded viral micropatch, various shape-coded microparticles are fabricated with carboxyl groups on the surfaces for specific immobilization of adenoviral vectors. The chemical functionalization is achieved by the incorporation of acrylic acid to photocurable polymer solution. Then, two adenoviral vector immobilization methods are developed with this shape-coded microparticle. The first method is to directly link the carboxyl groups on the microparticle and the primary amine groups on the surface proteins of adenoviral vectors using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) plus N-hydroxysulfosuccinimide (Sulfo-NHS) crosslinking reaction. The immobilization of adenoviral vectors in this approach is confirmed by an immunofluorescence test and a scanning electron microscope (SEM) observation. The second method utilizes an avidin-biotin interaction. In this approach, both the microparticles and adenoviral vectors are biotinylated using amine-activated and amine-reactive biotin reagents, respectively. Then, they are linked by the mediation of avidin. The immobilized adenoviral vectors are well observed using a SEM.
The localized viral gene delivery of two types of the encoded viral micropatches is evaluated by transducing a human osteosarcoma cell line (U-2 OS) cultured in a standard 96-well microtiter plate. The first type of the encoded viral micropatch fabricated via EDC/Sulfo-NHS reaction shows low rate of the localization of gene delivery due to an escape of non-specifically bound adenoviral vectors. However, the second type of the encoded viral micropatch fabricated utilizing avidin-biotin interaction offers highly localized gene delivery. This is owing to the viral receptor-independent transduction of the biotinylated adenoviral vector, which is further supported by the transduction experiment of an adenovirus receptor-deficient cell line.
Finally, I demonstrate a multiplexed GPCR internalization assay based on the localized gene delivery with the encoded viral micropatches. The development of high-throughput cell-based GPCR functional assays is very important for screening large compound libraries in the drug discovery process and ligand-induced receptor internalization assays have broad applicability to various GPCR subfamilies among several GPCR assay formats. However, high-content imaging is required for fluorescence-based intracellular measurement of receptor internalization. To address this issue, I fabricate three types of encoded viral micropatches with adenoviral vectors bearing green fluorescence protein (GFP)-tagged GPCR genes. Then, the responses of multiple GPCRs against one ligand treatment is acquired in one reaction site by achieving simultaneous expression of multiple GPCRs with the fabricated viral micropatches in a cell monolayer cultured in a well of a 96-well plate. High-content analysis of this micropatch-based multiplexed assay shows comparable results in the receptor internalization with the conventional singlet assay using free adenoviral vectors while reducing the number of pipetting actions.