Manipulation of Gene Expression in Mouse Pancreas via Intraductal Delivery of Adeno-Associated Viral Vectors
利用胰管内递送腺相关病毒载体调控小鼠胰腺中的基因表达
(*contributed equally to this work) 发布: 2026年04月20日第16卷第8期 DOI: 10.21769/BioProtoc.5657 浏览次数: 469
评审: Gururaj RaoAnonymous reviewer(s)
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参见作者原研究论文
The authors used this protocol in:
Dec 2018
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Abstract
The rising global incidence of pancreatitis, pancreatic cancer, and diabetes has increased the need for efficient in vivo gene manipulation approaches to study the pancreas and develop new therapies. Although transgenic mouse models are widely used, they are time-consuming and costly to generate and maintain. Systemic viral delivery methods offer greater flexibility but often lack pancreatic specificity and require high viral doses. Here, we describe a streamlined protocol for intrapancreatic ductal delivery of adeno-associated viruses (AAVs) for targeted gene delivery. Our protocol requires standard surgical equipment and can be implemented in most laboratories. Specifically, we adopted a clamping strategy at the hepatopancreatic duct near the liver, as well as beneath the major duodenal papilla at the duodenum. This strategy exposes the duodenal papilla, facilitating viral delivery, preventing backflow, and enabling efficient pancreatic transduction at lower viral doses. Overall, this method provides a fast, simple, and effective approach for pancreas-targeted gene manipulation, facilitating preclinical studies of pancreatic biology and disease.
Key features
• Rapid, pancreas-specific in vivo gene manipulation using simple rodent surgical techniques.
• Efficient gene manipulation can be achieved with lower viral doses while minimizing off-target effects.
• AAVs trigger minimal adverse complications, and the surgery is well-tolerated in mice.
• This method can be combined with traditional genetic manipulation and lineage tracing to enhance studies of gene function or pancreatic diseases.
Keywords: Intrapancreatic ductal injection (胰管内注射)
Gene delivery (基因递送)
Adeno-associated virus (腺相关病毒)
In vivo islet transduction (体内胰岛转导)
Aseptic mouse surgery (小鼠无菌手术)
Graphical overview
Schematic illustrating the murine hepatopancreatic duct injection site and the experimental timeline. (A) Anatomical location of the major duodenal papilla, and the placement of clamps and the injection needle. (B) Surgery timeline.
Background
The pancreas is located in the upper abdomen, behind the stomach, and contains both exocrine and endocrine compartments. The exocrine pancreas comprises approximately 90% of the organ and produces digestive enzymes such as amylase, lipase, and proteases [1]. These enzymes are transported through a branched pancreatic ductal system that joins the bile duct to form a hepatopancreatic duct, which empties into the duodenum through the major duodenal papilla to aid food digestion [2]. In contrast, the endocrine pancreas accounts for only 1%–2% of the pancreatic mass and is organized into clusters known as islets of Langerhans. Islet cells secrete hormones, including insulin and glucagon, that regulate blood glucose levels [3]. Disorders such as chronic pancreatitis, cystic fibrosis, or pancreatic duct obstruction by tumors or cysts can result in exocrine pancreatic insufficiency and malnutrition, whereas obesity and diabetes place a significant burden on endocrine islet hormone production and secretion [2].
With the global rise in pancreatic disorders, faster development of effective therapies is urgently needed [2,4,5]. Preclinical rodent models provide powerful systems for modeling disease etiology and evaluating novel therapeutic strategies. For example, Kras and Trp53 mutant mice recapitulate key features of pancreatic ductal adenocarcinoma, while non-obese diabetic (NOD) mice exhibit accelerated autoimmune-mediated islet destruction that parallels type 1 diabetes [6]. However, generating and maintaining transgenic mouse models is complex and time-consuming, requiring large colonies to preserve specific genotypes [7]. To overcome these limitations and enable rapid, flexible gene manipulation in the pancreas, injectable transduction methods via viral vectors have been developed. Viral vector-mediated gene manipulation can incorporate regulatory elements to enhance cell specificity and increase transgene expression. A variety of viral vector systems have been explored for this purpose, including retroviruses, lentiviruses, adenoviruses, herpes simplex viruses, and adeno-associated viruses (AAVs). Among these, AAVs offer relatively low immunogenicity and have been extensively validated for their safety and long-term transgene expression in a wide range of animal models of human disease [8].
In this protocol, we demonstrate intrapancreatic ductal delivery of AAVs for islet-specific gene manipulation. This procedure relies on standard rodent surgical equipment and can be rapidly implemented in most laboratories. In contrast to intravenous and intraperitoneal injections, direct AAV injection into the hepatopancreatic duct via the duodenal papilla allows viral vectors to rapidly diffuse throughout the pancreas, enabling efficient transduction of exocrine and endocrine cells with minimal off-target effects. As a result, comparable transduction can be achieved with lower AAV doses than with systemic delivery. Our technique builds on previously described intraductal injection methods to enhance efficiency and ease of use [9,10]. Specifically, the protocol employs a faster, catheter-free injection, reducing surgical time and complexity without compromising transduction quality or efficiency. Injection is facilitated by clamping beneath the duodenal papilla to create a clear injection site and prevent backflow into the duodenum. Together, these refinements enable faster surgeries while maintaining efficient pancreatic transduction.
Materials and reagents
Biological materials
1. Adeno-associated virus, 1011 genome copies/mouse
Note: In this manuscript, we used scAAV8-Ins1Cre [11] to induce beta cell–specific gene recombination and used scAAV8-Ins1Empty as control. Both vectors are custom-ordered from Vector Biolabs (pAAV210119-1011afm and VB230512-1203mkx, respectively). Increased AAV dosage can be used but may lead to off-target effects with little increase in transduction efficacy.
2. 6–8-week-old mice
Notes:
1. Young adult mice are recommended, as their duodenal papillae and hepatopancreatic ducts are fully developed. Papilla size and duct thickness vary with age, strain, and sex. In our experience, NOD mice have more prominent papillae than C57BL/6J, with optimal surgical outcomes observed in NOD mice > 6 weeks and C57BL/6J mice > 8 weeks. Ten-week-old CD1-mice are also suitable [12], and intrapancreatic ductal delivery of substrates via the cystic duct has been performed in 4-week-old FVB/N mice [13]. Older mice (>16 weeks) can be used; however, increased adiposity can make it difficult to visualize the hepatopancreatic duct in the surgical field, and age-related fibrosis or inflammation may partially limit AAV delivery within the pancreas.
2. In this study, we used 6-week-old female NOD.Cpefl/fl.ROSAmTmG mice [Cpetm1a(EUCOMM)Hmgu Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J].
Reagents
1. General inhalation anesthetic isoflurane (Fresenius Kabi, catalog number: M60303)
2. Pharmaceutical-grade sterile 0.9% NaCl (B. Braun, catalog number: L8002)
3. Dulbecco’s phosphate-buffered saline (DPBS) (Gibco, catalog number: 14190144)
4. Ophthalmic lubricant (Optixcare®) (Aventix, catalog number: B07CQ9MN9Y)
5. Meloxicam injectable (Metacam®) (Boehringer Ingelheim, catalog number: NDC 0010-6013-01)
6. Buprenorphine injectable (Temgesic®) (controlled substance; order through your institutional animal care facility)
7. Chlorhexidine skin disinfectant 4% (Stanhexidine®) (Omega Laboratories, catalog number: L0000014)
8. Pluronic F-68 non-ionic surfactant (Gibco, catalog number: 24040032)
9. Fast green dye (Sigma, catalog number: F7252)
10. Histoacryl tissue glue (B. Braun, catalog number: 1050052)
11. DietGel recovery enhanced water gel (ClearH2O, catalog number: 72-06-5022)
12. Ethyl alcohol 70% (Commercial Alcohols, catalog number: P016Ea95)
13. Prevail concentrate (Prevail, catalog number: 909-12305)
Solutions
1. AAV buffer (see Recipes)
2. AAV working solution (see Recipes)
3. Prevail working solution (see Recipes)
Recipes
1. AAV buffer
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Fast green dye | 0.1% | 10 mg |
| Pluronic F-68 non-ionic surfactant | 0.001% | 1 µL |
| DPBS | 100% | 10 mL |
Prepare AAV buffer and filter sterilize. This buffer can be stored at room temperature.
2. AAV working solution
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| AAV stock solution (~1013 vg/mL) | 1011 vg/mouse | 10 µL |
| AAV buffer | 90% | 90 µL |
Prepare AAV working solution in a Biosafety Level 1 biological safety cabinet dedicated to virus work. The calculation is based on transducing a single young mouse (25–30g body weight) with AAV8, a serotype known for its pancreatic tropism [14]. It is advisable to produce or purchase self-complementary adeno-associated virus (scAAV) stocks with titers of at least 1013 viral genomes (vg)/mL. Viral dosage varies depending on the vector and gene of interest; therefore, pilot testing is recommended for each project to determine optimal conditions. AAV working solution should be freshly prepared and kept on ice prior to mouse surgery.
3. Prevail working solution
| Reagent | Final concentration | Quantity or volume |
|---|---|---|
| Prevail concentrate | 1× | 1 part |
| Tap water | 39 parts |
Prepare 1:40 working solution from Prevail concentrate; it is stable up to 30 days after dilution.
Laboratory supplies
1. 1 mL syringes (Air-Tite, catalog number: 14-817-179)
2. Insulin syringe (Air-Tite, catalog number: 76290-414)
3. 30 G needles (BD, catalog number: 305106)
4. 25 G needles (BD, catalog number: 305122)
5. 4-0 absorbable monofilament sutures (Ethicon, catalog number: Y513G)
6. 5-0 absorbable braided filament sutures (Ethicon, catalog number: J463G)
7. 100 mm Petri dishes (ESBE Scientific, catalog number: SPL-10090)
8. 4 × 4 gauze tissue (10 per surgery) (Safe Dent, catalog number: C6012)
9. 2 × 2 gauze tissue (10 per surgery) (Safe Dent, catalog number: C6011)
10. 6-inch cotton tipped swabs (10 per surgery) (Puritan, catalog number: 806-WC)
11. Autoclave pouches (Fisher Scientific, catalog number: 01-812-50)
12. 10 μL graduated pipette tips (Labcon, catalog number: 1038-260-000-9)
13. Surgical gloves (Ansell, ENCORE® Latex Acclaim)
Equipment
1. Large loop scissors (FST, catalog number: 14101-14)
2. Sharp tip scissors (FST, catalog number: 14002-12)
3. Blunt tip scissors (FST, catalog number: 14013-17)
4. Toothed micro-Adson forceps (FST, catalog number: 11019-12)
5. Serrated micro-Adson forceps (FST, catalog number: 11018-12)
6. Ring forceps (FST, catalog number: 11103-09)
7. Micro needle holder (FST, catalog number: 12500-12)
8. Colibri retractors (FST, catalog number: 17000-04)
9. Straight clip (ROBOZ, catalog number: RS-5452)
10. Curved clip (ROBOZ, catalog number: 5459)
11. Cordless small pet hair grooming trimmer (Oneisall, catalog number: N6)
12. Anesthetic machine (Dispomed, Moduflex System)
13. Ear notch (FST, catalog number: 24214-02)
14. V-shaped heating bed with microflex breather (E-Z systems, catalog number: HB-1000-V)
15. Hot-bead sterilizer (preheated to 250 °C) (FST, catalog number: 18000-45)
16. Dissection microscope with 0.5× Aux objective (Nikon, catalog number: MZ745T)
17. Ring light with dial (Nikon, catalog number: MXK60559)
18. Goose neck light source (Dolan-Jenner, catalog number: Mi-150)
19. Heating pad (Sunbeam, catalog number: 756-500-CNR)
20. Timer (Fisher, catalog number: 14-649-17)
21. Scale (Fisher, catalog number: 8343501667)
22. Trimmer (Oneisall N5, catalog number: CW03002)
Procedure
文章信息
稿件历史记录
提交日期: Jan 26, 2026
接收日期: Mar 2, 2026
在线发布日期: Mar 22, 2026
出版日期: Apr 20, 2026
版权信息
© 2026 The Author(s); This is an open access article under the CC BY-NC license (https://creativecommons.org/licenses/by-nc/4.0/).
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Soukhatcheva, G., Stanley, L., Dai, L., Komba, M., Andriiets, V., Johnson, J. D., Verchere, B. C. and Chen, Y. (2026). Manipulation of Gene Expression in Mouse Pancreas via Intraductal Delivery of Adeno-Associated Viral Vectors. Bio-protocol 16(8): e5657. DOI: 10.21769/BioProtoc.5657.
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分类
医学
微生物学 > 异源表达系统 > 腺相关病毒
生物科学 > 生物技术 > 动物外科手术
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