Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction
March 17th, 2008
The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.
The hair follicle is a complex skin appendage consisting of six concentric cylinders with several distinct cell types that produce highly specialized proteins. The continuous cycling of the hair follicle through its distinct growth stages makes the appendage suitable for targeted gene therapy, which can potentially correct baldness and loss of hair pigmentation. In this video, we demonstrate a method for the histo culture of hair, growing skin, and ex vivo transduction of hair follicles with green fluorescent protein.
This efficient transduction technique is useful for following the promoter activity of various genes of interest in various regions of the follicle throughout its growth cycle. My name is Robert Hoffman. I'm president of Anti-Cancer Inc.
Today we're going to show you very powerful procedures for hair follicle gene therapy. This protocol starts with the histo culture of skin, followed by collagenase treatment. First miser sacrifice six days after depletion, and the back skin is removed at the level of the subcu.
After removing the skin, the subcutaneous tissue is removed. The skin is then rinsed in calcium and magnesium free PBS. At this point, the dissected skin is transferred to media Following PBS rinsing.
Skin samples are cut to small pieces approximately one millimeter cubed. A fraction of the specimens are set aside and cultured in RPMI 1640 and 10%fetal bovine serum as untreated controls. The rest of the specimens are incubated in two mg per mil.
Collagenase solution. In RPMI medium for 45 minutes to 3.75 hours at 37 degrees digested specimens are then washed in calcium magnesium free PBS histo cultures can be infected with virus for GFP transduction. Now that collagenase has made the hair follicles more accessible to virus in order to induce hair follicles.
To express GFP, they are first infected with virus adenovirus. Carrying A GFP plasmid is then added to the collagenase treated skin. Histo cultures at 2.4 million to 5 billion platforming units per mil.
Following the addition of virus, they're now placed in the incubator for one to six hours after viral transduction of GFP in skin histo cultures and approximately 24 hours after harvest, the specimens can be grafted on either nude or C 57 black six.Mice. GFP expression can then be assessed using fluorescence microscopy performed either on histo cultures or visualized in vivo on grafted skin. In order to determine the efficiency of GFP transduction, the total number of hair follicles and GFP positive hair follicles are determined under bright field and fluorescence microscopy.
Five randomly chosen microscope fields covering an area of 0.581 millimeters squared are analyzed. For GFP expression, at least 500 hairs per specimen are counted to generate the percentage of GFP positive hair follicles. Hair follicles in which GFP is visualized anywhere in the hair bulb or shaft are scored as GFP positive.
We've just shown you a highly efficient method for the transduction of GFP and hair follicles, which will be extremely useful for gene therapy. Such knowledges of clinical importance because it has implications for curing alopecia or baldness, preventing chemotherapy induced alopecia, and permanently modifying hair color. Thank you for watching and good luck with your experiments.
Overview
The hair follicle is a complex skin appendage that undergoes continuous cycling, making it a target for gene therapy. This article discusses methods for histo culture and gene transduction in hair follicles, which may have implications for treating hair loss.
Key Study Components
Area of Science
- Neuroscience
- Gene Therapy
- Dermatology
Background
- Hair follicles consist of multiple cell types and structures.
- They cycle through growth stages influenced by stem cells.
- Gene therapy can potentially address conditions like baldness.
- Understanding hair follicle biology is crucial for effective treatments.
Purpose of Study
- To demonstrate a method for histo culture of hair follicles.
- To explore gene transduction techniques for hair follicles.
- To assess the efficiency of GFP expression in hair follicles.
Methods Used
- Histo culture of skin samples followed by collagenase treatment.
- Infection of hair follicles with adenovirus carrying GFP plasmid.
- Assessment of GFP expression using fluorescence microscopy.
- Analysis of transduction efficiency by counting GFP positive hair follicles.
Main Results
- Efficient transduction of hair follicles with GFP was achieved.
- GFP expression can be visualized in vivo and in histo cultures.
- The method shows potential for applications in gene therapy.
- Implications for treating alopecia and modifying hair color were discussed.
Conclusions
- The study presents a powerful method for hair follicle gene therapy.
- Understanding gene transduction can lead to advancements in treating hair loss.
- Future research may focus on clinical applications of these techniques.
