summary

The live electrical conversion technology of newborn mouse retina provides a key means for retinal development and disease research. The study utilized the Venet Gene Pulser 830 square wave electroporation instrument to optimize the electrical conversion parameters, achieve efficient delivery and expression of exogenous genes in the retina of newborn mice, significantly improve transfection efficiency, and provide a reliable technical platform for retinal related basic research and translational medicine.

Introduction

As a key tissue for visual signal transmission, the study of the developmental mechanism and disease occurrence mechanism of the retina has always been a hot topic in the field of ophthalmology. The retina of newborn mice is in a rapid development stage, and gene manipulation can effectively study the role of genes in retinal cell differentiation, migration, and functional establishment. However, traditional gene delivery methods have problems such as low efficiency and significant cell damage in in in vivo retinal manipulation, making it difficult to meet the needs of precise scientific research.

Electroconversion technology instantly changes cell membrane permeability through pulsed electric fields, allowing exogenous nucleic acids and other macromolecules to enter cells, demonstrating advantages in gene delivery in living tissues. The Gene Pulser 830 square wave electroporation device from Veneto provides an efficient and accurate solution for in vivo electroretinolysis in newborn mice with its advanced square wave pulse technology, intelligent monitoring system, and flexible parameter settings, helping researchers overcome traditional technological bottlenecks.

Materials and Methods

1. Experimental animals

C57BL/6J newborn mice born 0-2 days after birth (P0-P2) were provided with SPF grade animal rooms in our laboratory, with a rearing environment temperature of (22 ± 2) ℃, humidity of 50% -60%, and a 12 hour light/dark cycle.

2. Main reagents

Plasmid DNA (containing the target gene GFP, constructed in our laboratory), a certain reagent (used to prepare electroporation buffer containing NaCl, KCl, glucose and other components, pH adjusted to 7.4), anesthetic (isoflurane, used for mouse anesthesia), disinfectant reagent (75% ethanol).

3. Main instruments

Venet Gene Pulser 830 square wave electroporation apparatus, stereomicroscope (a certain brand, used for surgical observation), microinjection needle (outer diameter 10-15 μ m, used for plasmid injection), constant temperature heating plate (to maintain mouse body temperature).

Experimental Procedure

1. Anesthesia and fixation of mice

Place newborn mice in an anesthesia box containing isoflurane, induce anesthesia, and transfer them to a constant temperature heating plate to maintain stable body temperature. Gently fix the mouse's head and limbs with tape to ensure stable posture during the surgery.

2. Retinal localization and plasmid injection

Under a stereomicroscope, plasmid DNA containing the target gene (concentration of 1-2 μ g/μ L) was aspirated using a micropipette, and the needle was inserted into the temporal corneal edge of the mouse eye. Slowly insert the needle tip into the vitreous cavity, avoiding the lens, and accurately inject 5-10 μ L of plasmid solution into the subretinal space. During the injection process, pay attention to controlling the depth and speed of the needle to avoid damaging the retinal tissue.

3. Parameter setting and operation of electroporation

The Venet Gene Pulser 830 square wave electroporation instrument adopts a pre programmed optimization system, selects mammalian cell transfection mode based on the characteristics of newborn mouse retinal tissue, and calls the built-in recommended parameters of retinal cells: square wave pulse, voltage adjusted according to mouse age and eye size (generally 50-80V), pulse duration 10-20ms, pulse frequency 3-5 times, pulse interval 1-2s.

Gently clamp the mouse eyeball with the customized forceps type electrode (electrode spacing 1-2mm, surface treated with insulation to prevent current leakage), ensuring that the electrode is tightly attached to the eyeball surface and does not compress the eyeball. Starting the electroporation program through the foot switch, the instrument's 10 inch touch screen displays real-time pulse waveforms and parameter dynamics, making it easy for experimenters to monitor the entire process. During the process of electroporation, the instrument automatically performs intelligent impedance detection, pre pulse measures sample resistance, dynamically adjusts parameters, and ensures that each shock condition matches the state of retinal cells. At the same time, arc protection and polarity conversion technology eliminate arc damage, break through the cell membrane charge barrier, and improve the success rate of difficult to transfect cells.

4. Postoperative treatment

After the electrical conversion operation is completed, move the mouse back to the mother mouse's side and keep the environment warm and quiet. Observe the eye condition and survival of mice daily after surgery to ensure no infection or other complications occur.

Results and Discussion

Transfection efficiency detection

Three days after electroporation, mouse eyeballs were taken to prepare frozen sections of the retina, and the expression of green fluorescent protein (GFP) was observed under a fluorescence microscope. The results showed that obvious GFP fluorescence signals were visible in all layers of cells in the retina, especially in the ganglion cell layer, inner nucleus layer, and outer nucleus layer. The transfection efficiency was increased by more than 40% compared to traditional methods (based on internal testing data from Veneto). This is thanks to the high-intensity electric field of the Gene Pulser 830 square wave electroporation device from Venet, which instantly reshapes the cell membrane permeability and achieves efficient delivery of nucleic acid macromolecules.

Cell survival and tissue damage assessment

By observing the retinal tissue structure through hematoxylin eosin (HE) staining, the layers of the retina in the electroporation group of mice were clear and the cells were arranged neatly. Compared with the control group, there was no significant cell necrosis or inflammatory response. The Taipan Blue exclusion experiment showed that the survival rate of retinal cells after electroporation was over 90%, indicating that this electroporation technique causes minimal damage to retinal tissue and ensures the feasibility of subsequent experiments.

Comparative advantages with traditional technology

The traditional virus vector transfection method has problems such as long preparation cycle, high cost, limited vector capacity, and may trigger immune reactions. The in vivo electroporation technology mediated by the Gene Pulser 830 square wave electroporation instrument from Veneto does not require virus packaging and can directly deliver large molecules such as DNA, RNA, and proteins. It is easy and fast to operate, low in cost, and avoids potential risks of virus vectors. Its full waveform intelligent monitoring system ensures transparency and controllability of the experimental process, traceability of data, and assists in the accurate reproduction of scientific research results. The pre programmed optimization system has a built-in library of commonly used cell line parameters, which can be called with one click. It also supports custom pulse parameters and adapts to complex scenarios such as newborn mouse retina, demonstrating strong flexibility and applicability.

Technical optimization and precautions

During the experiment, it was found that the optimization of electroporation parameters is a key factor affecting transfection efficiency. High voltage or excessive pulse frequency may cause cell damage, while low voltage leads to insufficient transfection efficiency. The intelligent impedance detection function of the Gene Pulser 830 from Venete can dynamically adjust parameters based on sample resistance, effectively solving this problem. In addition, the concentration and volume of injected plasmids, the degree of electrode adhesion, and the age of mice all have an impact on the experimental results, and the experimental conditions need to be strictly controlled.

Application prospects

Basic research field

This technology can be widely applied to the functional research of genes related to retinal development, such as CRISPR gene editing technology combined with electroporation, which can achieve the knockout or insertion of specific genes in the retina, and study their roles in retinal cell differentiation and neural circuit formation; SiRNA delivery can be used to inhibit the expression of target genes and explore the mechanisms of genes in the occurrence of retinal diseases; In the research of stem cell reprogramming, electroporation technology can efficiently introduce reprogramming factors into retinal stem cells, providing a new approach for retinal regenerative medicine research.

Translational medicine field

In terms of live tumor electroporation, targeted therapy can be achieved for ocular tumors such as retinoblastoma by delivering therapeutic genes or drugs through electroporation; In vaccine development, electroporation technology can deliver antigen genes to retinal associated immune cells, induce immune responses, and provide new ideas for ocular vaccine research; In terms of in vivo gene therapy, for genetic retinal diseases such as retinitis pigmentosa and congenital melanoma, electroporation technology can introduce normal genes into retinal cells, laying the foundation for the clinical application of gene therapy.

Agriculture and Industry

Although the research focuses on the retina of newborn mice, the wide applicability of the Venet Gene Pulser 830 square wave electroporation device also makes it have great potential in the fields of agriculture and industry. In plant genetic improvement, it can be used for gene delivery in plant protoplasts to accelerate crop variety improvement; In the construction of yeast engineering bacteria and microbial metabolism engineering, electroporation technology can efficiently achieve exogenous gene introduction and improve the production efficiency of engineering bacteria.

With its innovative square wave pulse technology, intelligent monitoring system, and deep technical service support, the Gene Pulser 830 square wave electroporation instrument has become the trusted choice of over 500 top research institutions. Whether it is precise gene manipulation in basic research or innovative treatment exploration in translational medicine, this device provides an efficient and reliable technical platform for researchers. At present, Winide provides free technical solution consultation and product trial services to help researchers overcome the barriers of transfection technology and promote the development of life science research and related fields.

References

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