Eukaryotic CD36 Mutant Cell Line via Virus-Mediated Transfection
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Solution Overview
Problem
Current methods focus on prokaryotic cloning of CD36 genes, lacking a method for establishing eukaryotic cell lines stably expressing mutant CD36 genes, which is crucial for understanding CD36-related diseases and developing effective treatments.
Innovation Solution
A method involving RT-PCR, SOE-PCR, and virus-mediated transfection is used to create eukaryotic cell lines expressing mutant or normal CD36 genes, utilizing primers and vectors to ensure accurate gene expression and stability, allowing for the study of CD36 functions and disease mechanisms.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If prokaryotic cloning methods are used for CD36 gene research, then the cloning process is simple and quick, but the method cannot establish stable eukaryotic cell lines for studying CD36 functions and disease mechanisms
Solution Approach 1:
The patent uses a eukaryotic expression vector as an intermediary carrier to transport the mutant CD36 gene into eukaryotic cells. This vector system enables the gene to be expressed stably in eukaryotic cells, bridging the gap between simple cloning and complex eukaryotic expression requirements.
Solution Approach 2:
The patent replaces prokaryotic cloning mechanisms with eukaryotic expression mechanisms. By using eukaryotic cells and eukaryotic expression vectors, the system achieves stable gene expression and inheritance in eukaryotes, which is fundamentally different from prokaryotic cloning approaches.
2Ease of manufacture
If mutant CD36 genes are cloned in prokaryotes, then the cloning is straightforward, but stable expression and inheritance in eukaryotic cells cannot be achieved
Solution Approach 1:
The patent changes the fundamental parameters of the expression system from prokaryotic to eukaryotic. This involves using eukaryotic cells, eukaryotic expression vectors, and eukaryotic transcription/translation mechanisms to achieve reliable and stable gene expression and inheritance.
3Reliability
If eukaryotic cell lines are established for CD36 research, then stable expression and disease mechanism study are enabled, but the establishment process is complex and time-consuming
Solution Approach 1:
The patent performs preliminary actions by first constructing the eukaryotic expression vector with the mutant CD36 gene in vitro, then using this pre-prepared vector to transfect eukaryotic cells. This preliminary vector construction simplifies the subsequent cell line establishment process.
4Productivity
If prokaryotic cloning is used, then the process is fast and simple, but it cannot provide the platform for studying CD36 functions in human physiology and pathology
Solution Approach 1:
The patent creates a universal eukaryotic expression system that can express both normal and mutant CD36 genes in eukaryotic cells. This system serves multiple functions: studying CD36 protein structure, analyzing gene function, modeling disease mechanisms, and screening therapeutic drugs, making it highly versatile for various research applications.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach provides a stable platform for studying CD36-related diseases, drug development, and ensuring the safety of platelet transfusions by enabling the expression of CD36 proteins in eukaryotic cells, facilitating deeper insights into CD36's role in human physiology and pathology.
Implementation Method 1
amplifying the coding sequence (CDS) in CD36 mRNA by Reverse Transcription PCR (RT-PCR)
Implementation Method 2
splicing and amplifying the mutant CD36 gene and the EGFP fluorescent gene by SOE-PCR
Implementation Method 3
constructing and amplifying a MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector comprising the mutant CD36 gene and the EGFP fluorescent gene by ligating MT-CD36-EGFP to a pLV4/StripII-HIS10 vector
Implementation Method 4
transfecting the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector into the CHO-K1 cell line by using virus-mediated transfection of eukaryotic cells
Implementation Method 5
screening and constructing a eukaryotic cell line MT-CD36-CHO-K1 stablely expressing the mutant CD36 gene
Data Source
AI summary
A method for establishing eukaryotic expression cell line of CD36 mutant gene that encodes CD36 deficiency, the method including: (1) extracting total RNA from a whole blood sample derived from a CD36-deficient individual, and amplifying a coding sequence (CDS) in CD36 mRNA, to obtain a cDNA sequence fragment of the mutant CD36 gene; (2) splicing and amplifying the mutant CD36 gene and the EGFP fluorescent gene by SOE-PCR (Gene Splicing By Overlap Extension PCR) using four forward and reverse primers, to obtain a mutant gene fragment of MT-CD36-EGFP; (3) constructing and amplifying a MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector including the mutant CD36 gene and the EGFP fluorescent gene; (4) transfecting the MT-CD36-EGFP-pLV4/StripII-HIS10 eukaryotic expression vector into the CHO-K1 cell line by using virus-mediated transfection of eukaryotic cells.