Gene Overexpression - Gene Overexpression Techniques ()

Tissue-specific Cell Targeted Gene Manipulation: Gene expression

Gene expression refers to the process of synthesizing genetic information from genes into functional gene products. The products of gene expression are usually proteins, but the expression products of non-protein-coding genes such as transfer RNA (tRNA) or small nuclear RNA (snRNA) genes are functional RNAs.

Gene overexpression is to clone the coding region sequence (CDS) of the target gene into the corresponding plasmid or virus vector(like AAV vectors), and use the regulatory elements constructed on the vector backbone to enable the gene to achieve a large amount of transcription and translation under artificial control conditions, so as to achieve the overexpression of the target gene. In addition, reporter genes can be selected for tracing or resistance genes for screening.

Commonly used viral vectors include adeno-associated virus vectors（AAV vectors）, lentiviral vectors, adenoviral vectors, and retroviral vectors, which are widely used in in vitro and in vivo gene function studies.

Comparison of 4 commonly used viral vectors

Choose different vectors according to different experimental requirements, target genes and uses.

	Adenovirus	LV	Retrovirus	AAV vectors
Diameter	60-90 nm	90-110 nm	90-100 nm	20-30 nm
Copy type	independent	none	none	none
Gene capacity	5~6 kb	4 kb	1.6 kb	2.5 kb
Integration Mechanism	non-integrated	random with high frequency	random with high frequency	directional with low frequency
Cell infection	Wide range of types	Wide range of types	split cell	serotype determination
Titer range	10^11~12 pfu/ml	10^8~9 tu/ml	10^7~8 tu/ml	10^12~13 vg/ml
In vitro infection	available	recommended	available	available
Positioning injection	available	available	recommended	recommended

Virus type	Starting time	Duration	Titer requirements	Diffusion ability	Immunogenicity	Cell type
Adenovirus	2-3 days	＜10 days	＞1X10¹⁰	strong	strong	wide
LV	3-4 days	＞2 months	＞1X10⁸	normal	normal	wide
Retrovirus	3-4 days	＞2 months	＞1X10^7~8	normal	nornal	split cell
AAV	2-3 weeks	＞3 months	＞1X10¹²	strong	weak	serotype determination

Regulatory element selection

Category	Name	Function
Promoter	CMV, EF1a, hSyn, TRE3g, etc.	Broad-spectrum, tissue-specific, and inducible promoters initiate transcription
Common tags	GFP, EGFP, YFP, mCherry, DsRed2, tdTomato, BFP-tag, CFP, etc.	Fluorescent label
Common tags	His, flag, GST, HA, Myc, etc.	Affinity tag
Enhancer	WPRE, SV40, etc.	Enhance gene transcription
Resistance gene	KanR, AmpR, etc.	Prokaryotic resistance screening
Resistance gene	PuroR, Neo, Diphtheria toxin, Actidione, etc.	Eukaryotic cell resistance screening
Connection element	2A（T2A, P2A, E2A, etc.), IRES	Used to connect 2 expressed genes

The characteristics and adaptability of the three types of overexpression AAV vectors

1 Target protein expression

A series of modifications such as fusion or non-fusion fluorescent tags, affinity tags, and subcellular localization series can be added to protein expression at the same time as conventional protein expression.

Example graph structure:

2 Non-coding RNA expression

Different from conventional protein expression, since non-coding RNA (link non-coding RNA introduction) is only transcribed but not translated, no protein is produced, so such expression vectors (especially lncRNA) are best to express fluorescent proteins independently, and the modification of conventional protein expression is basically not available. At present, the common non-coding RNAs are mainly three types of expression vectors: microRNA, lncRNA, and circRNA.

Example graph structure: