Phenotype Analysis and Evaluation

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Cyagen provides a variety of evaluations for our customers, including molecular, cellular (in vitro), and animal (in vivo) phenotypic testing services. Our services span the field of gene therapy, covering multiple diseases such as those affecting neurological, cardiovascular, and cerebrovascular systems, tumors, and more. We provide customers with a comprehensive phenotype analysis system service platform to support every research model project.

Phenotype Analysis for Gene Therapy:

Type	Technology
Immunological Detection	Western Blot (WB) detection
	Immunohistochemistry (IHC) detection
	Immunofluorescence (IF) detection
	ELISA detection
	Fluorescence–activated cell sorting (FACS)
Physiological and Biochemical Testing	Small animal ultrasound imaging
	Blood biochemical index detection
	ECG detection of rats and mice
	Blood Pressure Check
Behavioral Testing	Morris water maze task
	New object recognition
	Gait detection
	Grip test
	Open field test
	Swim test
Cell Function Test	Cell proliferation assays
	Apoptosis and cell cycle assays
	Cell migration & Invasion assays
Molecular Biology Testing	Real-time fluorescence-based quantitative PCR
Phenotype Analysis of Other Diseases	In vivo imaging, Dyeing experiments, etc.

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AAV Viral Vectors - in Vivo Injection

In practical applications, different target organs require different AAV injection methods. Before injection, we need to clarify the injection dosage, injection method, operation method, etc. Different injection strategies have a great impact on the final therapeutic effect. Based on years of experience in animal experiment operation platforms, Cyagen can provide intravenous injection or orthotopic injection of different tissues. Since different serotypes match the recognition and infection capabilities of different tissue types, we recommend that you use a suitable injection method to help your experiment achieve credible results.

Common Injection Methods for Different Tissues

Tissue type	Injection method	Tissue type	Injection method
Liver	Tail vein injection; Liver parenchymal injection	Skin	Hypodermic injection
Heart	Aortic injection; Intramyocardial fixed point injection	Intestine	Tail vein injection; Mesenteric artery injection; Intestinal fixed
Kidney	Tail vein injection; Renal vein injection	Adipose	Tail vein injection; Intraperitoneal injection
Blood vessel	Tail vein injection; Vascular clipping injection	Spinal cord	Local spinal cord injection; Intrathecal injection
Lung	Tail vein injection; Intratracheal injection; Nasal drip	Brain	Tail vein injection; Intraventricular injection; Brain stereotactic injection
Muscle	Local fixed point injection; Tail vein injection	Eye	Intravitreal injection
Joint	Joint cavity injection	Testis	Orthotopic injection

Common Problems of AAV in Vivo Injection

Q1: The injection method of AAV virus includes tail vein injection and local orthotopic injection. How should we choose?

The in vivo injection (administration) of AAV includes systemic administration and local administration. Systemic administration injection sites mainly include the tail vein, jugular vein, orbital vein, and abdominal vena cava; common local administration sites include stereotactic brain injection, intramuscular injection, orthotopic injection of myocardium, intravitreal injection, joint cavity injection, etc. The characteristic of systemic administration is that the volume of virus injection is large, requiring a volume of 100~1000μl. Its operation is relatively simple, the administration is convenient, and the virus spreads in the body in a wide range.
For local administration, the virus injection dose requires less, generally within 100μl, but the titer is higher; because the AAV is directly delivered to the target tissue, the local administration will have better targeting. For clinical gene therapy, the dose of virus injected by local administration is small, so the pathological toxicity will be smaller.

Q2: How long does the AAV take effect after injection? Is there a way to shorten its expression time?

After AAV enters the cell, it undergoes a process from single-stranded DNA (ssDNA) to double-stranded DNA (dsDNA). The time required for the target gene expression to become detected is 1 week or more depending on the target gene, since different genes have different peak expression times. Using Self-complementary Recombinant Adeno-Associated Virus (scAAV) can speed up expression. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, scAAV will modify one of the ITRs so that the two complementary halves of scAAV will associate to form one double stranded DNA (dsDNA) unit that is ready for immediate replication and transcription. The caveat of this construct is that instead of the full coding capacity found in rAAV (4.7-6kb), scAAV can only hold about half of that amount (≈2.4kb). Therefore, it can only be used for research with small gene fragments.

Q3: What is the purpose of injecting and purifying AAV in vivo?

If the virus is used to infect cells in vitro, it generally does not need to be purified, but it is necessary to purify any virus used for in vivo injection, whether it is a lentivirus or AAV. The reason is that in the packaging and production process of the virus, the unpurified virus liquid contains a large amount of virus shell, cell (293T) endotoxin, broken particles, and cell debris, etc. If these substances are injected into the animal body with the virus, it may cause a strong immune response, thereby affecting the survival rate of animals. In addition, virus purification is also a concentration process, which is conducive to subsequent dilution and dosing administration.

Inquiries and Quote Requests

Request a quote now. Alternatively, you can always email animal-service@cyagen.com or call 800-921-8930 to inquire about our services or obtain a quote for your project.

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