Lactotransferrin Gene Expression via Promoter Insertion
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Solution Overview
Problem
Current methods for increasing lactotransferrin gene expression in mammary glands are inefficient, limiting the production of this important protein in animal milk, which has wide-spectrum anti-bacterial, anti-oxidation, and anti-cancer properties.
Innovation Solution
A method involving gene editing to create a new gene combination by inserting the promoter of specific genes (αS1 casein, β casein, αS2 casein, κ casein, β-lactoglobulin, or α-lactalbumin) upstream or downstream of the lactotransferrin gene, using tools like CRISPR/Cas systems to generate DNA breaks and promote lactotransferrin expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used to increase lactotransferrin gene expression, then some expression increase may be achieved, but the efficiency is insufficient and expression levels remain low
Solution Approach 1:
The gene editing method divides the lactotransferrin gene structure into separate functional elements (promoter region, coding region, 3' UTR) and independently optimizes each segment. The promoter is replaced with strong constitutive promoters, the coding region is optimized for codon usage, and the 3' UTR is enhanced for mRNA stability, allowing systematic improvement of expression levels
Solution Approach 2:
The patent applies multiple parameter changes to the gene structure including: replacing the native promoter with strong constitutive promoters (CMV, SV40, Rous Sarcoma Virus), optimizing codon usage for the host organism, adjusting GC content, and modifying the 3' UTR sequence to enhance mRNA stability and translation efficiency, thereby dramatically increasing expression levels
2Productivity
If gene editing tools are used to create new gene combinations, then lactotransferrin expression can be significantly increased, but the complexity of the gene editing process increases
Solution Approach 1:
The patent uses CRISPR-Cas9 system as an intermediary tool to facilitate precise gene editing. The guide RNA (gRNA) acts as a mediator that directs the Cas9 nuclease to specific target sequences in the lactotransferrin gene, enabling targeted modifications without requiring complex homologous recombination protocols or multiple plasmid systems
Solution Approach 2:
The patent replaces traditional mechanical gene manipulation methods (such as restriction enzyme digestion, ligase-based cloning, and electroporation of large DNA constructs) with a more efficient molecular biology approach using CRISPR-Cas9 nucleases and base editing systems, which can achieve gene modifications through programmable RNA-guided mechanisms rather than physical DNA manipulation
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 significantly increases lactotransferrin expression levels in mammary glands, leading to higher protein content in animal milk, providing enhanced nutritional and health benefits.
Implementation Method 1
DNA breaks are generated simultaneously at three different specific sites in the genome of an animal cell
Data Source
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AI summary
The present invention relates to technical fields of genetic engineering and bioinformatics, and particularly relates to a method of specifically increasing lactotransferrin gene expression levels and an application thereof. The method adopts the promoter of the specific gene, which is selected from αS1 casein gene, β casein, αS2 casein, κ casein, β-lactoglobulin or α-lactalbumin genes, to drive the LTF gene expression, thereby leading to up regulation of the LTF gene expression level in mammary glands and having great application potential in increasing the lactotransferrin content in animal milk.