Lentivirus Packaging

Efficient transfection of viruses

Ubigene provides lentivirus, adenovirus and adeno-associated virus (AAV) with different sizes and grades of purification. Our products can easily achieve knockout (KO), overexpression and knockdown in vitro and in vivo.

Comparison of different viruses:

Type	Lentivirus	Adenovirus	AAV
Origin	HIV-1	Adenovirus type 5	AAV2
Genome	Two linear and continuous single-stranded RNAs	A linear and continuous double-stranded DNA	A linear and continuous single-stranded DNA
Genome size	~9.2 kb (WT)	~36 kb (WT)	~4.7 kb (WT)
Expression Duration	long-term stable expression	Transient expression	Long-term expression
Integrate to cell genome	Yes	No	Low frequency
Immunogenicity	Low	High	Very low
Capacity	~6.4 kb	~8.3 kb	~4.7 kb
Advantages	· Wide range of applications; · High transduction efficiency; · stable hereditary.	· High transduction efficiency; · Large capacity; · Expandable.	· Low immunogenicity; · High safety; · High specificity.

Lentivirus Packaging Technical Details

The lentivirus vector is based on HIV-1 (Human Immunodeficiency Virus-1) and developed from herpes virus VSVG capsid. It is a pseudovirus, as its pathogenic genes will be deleted and replaced by exogenous target genes. Lentiviruses have a wide spectrum of infections and can effectively infect divisive and non-divisive cells. Exogenous genes can be effectively integrated into host chromosomes by Lentiviruses, to achieve continuous expression. Thus, lentivirus has become a useful tool for introducing exogenous genes. Lentiviral systems have been widely used in various cell lines for gene knockin/knockout, overexpression, RNA interference, microRNA related researches and in vivo experiments.

Product Specs

Standard	Titer	Turnaround	Application
Standard	1*10^8 TU	2-3weeks	Cell transduction
Large size	1*10^9 TU	2-3weeks	Cell transduction
Ultra-purified	1*10^9 TU	2-3weeks	In vivo injection

Work Flow and Validation：

FAQ about lentivirus, click to show

·FAQ about lentivirus:

1. How long does it take for gene expression after transduction?

It takes a long time for expression after lentivirus transduction. But fluorescence can be observed 24 hours after transduction on the cells with strong metabolism (such as 293T, HEK293, etc.); the fluorescent protein expression of cells with slow metabolism (such as primary culture cells, neural stem cells, embryonic stem cells, etc.) takes a longer time, i.e. 72-96 hours or even longer after transduction. The transduced cells can be continuously cultured for one week, and the effect of transduction of the virus to the target cells can be determined by observing the expression time and intensity of fluorescence.

2. How to improve transduction efficiency?

Ensure that the cells are in good growth condition before transduction. Use 293T for titer test to verify whether the titer of virus drops significantly; properly increase the MOI and extend the transduction time.

3. After lentivirus transduction, the condition of cells is very poor or even a large number of deaths. How to solve this problem?

Poor cell conditions before transduction, exceeding MOI, and cytotoxicity of target genes may lead to abnormal cell conditions.The main solutions are:
(1) make sure the pre-transduction cell condition;
(2)reduce the MOI to verify the cytotoxicity of the target gene;
(3) observe the cells after 6 hours of transduction, change the fresh medium in case of abnormality of cells, if not, change the medium 24 hours later.

Knockout lentivirus

The lentivirus knockout plasmids would be packaged as lentiviruses.After the cells are infected with lentiviruses, it can stably express gRNA and Cas9 protein to knockout the target gene.

Knockout lentivirus vector selection:

YKO series	Vector	Reporter gene; Selection marker
Lentiviral YKO plasmid	YKO-LV001-single-gRNA	EGFP/mCherry,Puro/Neo
Lentiviral YKO plasmid	YKO-LV001-dual-gRNA	EGFP/mCherry,Puro/Neo
Lentiviral Cas9 plasmid	YCas-LV001	EGFP
Lentiviral Cas9 plasmid	YCas-LV002	Hygro

Because of the limitation of lentivirus capacity, the Cas9 protein, which is ~4kb, should be packed separately from gRNA. Generally, the two lentiviruses are used to co-infect the host cells.

Expression lentivirus

The gene expression plasmid would be constructed and packaged as lentivirus. After infecting host cells with overexpression lentivirus, drug resistance carried by virus vector can be used for drug screening, so as to obtain stable cell lines expressing target genes. Lentivirus can be used in vitro (including cell lines, immortalized cells, primary cells, and stem cells) and in vivo.

Gene overexpression lentivirus vector selection：

YOE series	Vector	Reporter gene; Selection marker
Lentiviral YOE plasmid	YOE-LV001	EGFP/Puro
	YOE-LV002	mCherry/Puro
	YOE-LV003	EGFP/Neo
	YOE-LV004	mCherry/Neo
	YOE-LV005	Puro
	YOE-LV006	EGFP
	YOE-LV007	mCherry
	YOE-LV008	Luciferase/Puro

Knockdown lentivirus

The shRNA expression vector would be constructed and packaged as lentivirus. After infecting host cells with lentivirus, drug resistance carried by virus vector can be used for drug screening, so as to obtain stable cell lines expressing shRNA. Lentivirus can be used in vitro (including cell lines, immortalized cells, primary cells, and stem cells) and in vivo.

Gene interference lentivirus vector selection ：

YSH series	Vector	Reporter gene; Selection marker
Lentiviral YSH plasmid	YSH-LV001-shRNA	EGFP/Puro
	YSH-LV002-shRNA	mCherry/Puro
	YSH-LV003-shRNA	EGFP/Neo
	YSH-LV005-shRNA	EGFP/Hygro
	YSH-LV004-shRNA	mCherry/Neo
	YSH-LV006-shRNA	Puro
	YSH-LV007-shRNA	EGFP
	YSH-LV008-shRNA	mCherry
	YSH-LV009-shRNA	Luciferase/Puro