make pigeons sanitary - Your Published Bio Team Output
**Pre-Project**To achieve the goal of making pigeons sanitary, we could look at modifying the endogenous pathway involved in pigeon gut microbiota. Pigeons are known to carry various pathogens on their feathers and in their droppings, which can lead to the spread of diseases. By modifying their gut microbiota, we could potentially reduce their ability to carry harmful pathogens.
One pathway that could be imported from a different species is the bacteriophage therapy pathway. Bacteriophages are viruses that can infect and destroy specific bacteria, without harming other bacteria or human cells. By importing bacteriophage therapy from a species of bacteriophage that targets the bacteria commonly found in pigeon droppings, we could reduce the number of harmful bacteria present in their environment.
To implement these modifications, we could investigate the use of probiotics to modify the pigeon gut microbiota. By introducing beneficial bacteria into the pigeon's diet, we could promote the growth of microbiota that do not carry harmful pathogens. Additionally, we could investigate the use of engineered bacteriophages to selectively target harmful bacteria found in pigeon droppings, thus reducing the overall risk of disease transmission.
It is important to note that any modifications made to pigeons should be done with consideration for ethical concerns, and potential negative impacts on the ecosystem. We should also consider the potential long-term effects of these modifications, and whether they could unintentionally harm the pigeons or other organisms in their environment.
**Genes:** 1. CRISP/Cas-9 gene editing of the microbiota: The microbiota of pigeons plays a crucial role in the maintenance of their health. By using CRISPR/Cas-9 gene editing technology, we can target and modify specific bacteria present in the microbiota. For example, we could target bacteria that are known to cause disease and modify them to become non-pathogenic.
2. RNAi knockdown of pathogenic genes: RNA interference (RNAi) is a technique used to silence specific genes. By using RNAi, we can knock down the expression of genes that are involved in pathogenesis in pigeons. For example, we could target genes that are responsible for the virulence of certain bacteria or viruses present in pigeons.
3. CRISPR/Cas-9 gene editing of immune genes: The immune system of pigeons plays a critical role in their defense against pathogens. By using CRISPR/Cas-9 gene editing, we can modify immune genes to make them more effective in fighting off infections. For example, we could modify genes that code for antimicrobial peptides, which can actively kill pathogens.
4. RNAi knockdown of odor-causing genes: One of the biggest concerns with pigeons is the odor associated with their droppings. By using RNAi, we can knock down the expression of genes that lead to the production of odor-causing compounds. For example, we could target genes involved in the metabolism of sulfur-containing compounds to reduce the sulfuric odor associated with pigeon droppings.
5. CRISPR/Cas-9 gene editing of genes involved in feather production: Pigeon feathers can contribute to the spread of disease, as they can harbor pathogens. By using CRISPR/Cas-9 gene editing, we can modify genes that are involved in feather production, resulting in feathers that are less likely to harbor pathogens. For example, we could modify genes involved in the expression of keratin, a protein that makes up feathers, to make them more resistant to bacterial colonization.
**Regulatory Elements:** Promoters:
1. The promoter sequence for the Ubiquitin C gene can be an ideal promoter for gene editing and knockdown techniques in pigeons. This promoter is highly active and ubiquitous, meaning it can drive high levels of expression in a wide range of tissues, including the immune system and microbiota. Using this promoter can lead to efficient modification of microbiota, immune genes, and odor-producing genes.
2. The promoter sequence for the beta-actin gene can be useful for gene editing of feather production genes. This promoter has been shown to drive high levels of expression in feathers and can be used to modify genes involved in feather keratin expression and composition.
3. The promoter sequence for the Mitochondrial ATP Synthase Subunit 6 gene can be an ideal promoter for gene editing of the microbiota. This promoter is highly active in mitochondria, making it ideal for targeting bacteria within that organelle.
1. The IgM gene enhancer can be useful for enhancing the expression of immune genes in pigeons. This enhancer is active in B cells and can be used to drive high levels of expression of modified antimicrobial peptides.
2. The Lcn2 gene enhancer can be used to enhance the expression of natural antimicrobial peptides in pigeons. This enhancer regulates the expression of lipocalin 2, which is an important antimicrobial protein in mammals and could potentially function similarly in pigeons.
3. The beta-globin gene enhancer can be used to enhance the expression of genes involved in feather production. This enhancer is active in erythroid cells, which produce hemoglobin for the blood. However, it has also been shown to drive high levels of expression in the skin, providing a possible method for enhancing feather production genes.
1. The beta-globin gene terminator can be useful for gene editing of feather production genes. This terminator has been shown to be highly efficient in erythroid cells and can be used to terminate transcription of modified feather production genes.
2. The bovine growth hormone terminator can be used to terminate transcription of modified genes in the microbiota. This terminator has been shown to be efficient in various organisms, including bacteria, making it a good option for CRISPR/Cas-9 gene editing of microbial genes.
3. The human alpha-globin gene terminator can be used to terminate transcription of modified odor-producing genes in pigeons. This terminator has been used in various gene therapy clinical trials, and its efficiency has been demonstrated, making it an ideal choice for knocking down odor-producing genes.
**Vector & Delivery:** For the desired genetic modifications in pigeons to make them sanitary, I would recommend using the CRISPR/Cas-9 gene editing technique to modify the microbiota, immune genes and feather production genes in a targeted manner. To deliver the genetic modifications, I suggest using a viral vector, specifically the adeno-associated virus (AAV) vector for its ability to infect both dividing and non-dividing cells, its low immunogenicity, and long-lasting expression of the gene of interest.
For the modification of microbiota, the use of the Ubiquitin C promoter and the Mitochondrial ATP Synthase Subunit 6 promoter can be optimal in driving high expression in the microbiota. Also, the use of the bovine growth hormone terminator can ensure efficient transcription termination in bacteria.
For the modification of immune genes, the use of the IgM gene enhancer can be useful and efficient in driving high expression of modified antimicrobial peptides in B cells. The Ubiquitin C promoter can also be used to drive high expression in the immune system.
For the modification of feather production genes, the beta-actin promoter and beta-globin enhancer can be used to drive high expression in feathers. Furthermore, the beta-globin terminator can efficiently terminate transcription of the modified feather production genes.
Based on these choices, using the AAV vector for delivery can offer efficient and targeted delivery of the gene editing tools for a long-lasting effect in pigeons. The combination of these methods can help to optimize the desired genetic modifications to make pigeons sanitary.
**Selection Marker:** If a selection marker is deemed needed for this project we will use a fluorescent protein marker, such as GFP, to facilitate the selection and identification of successfully modified organisms. The fluorescent protein can be fused with the gene editing tools, allowing for easy identification of successful gene editing events in both in vitro and in vivo settings. Fluorescent microscopy can be used to easily visualize the expression of the fluorescent protein in tissues and cells, allowing for confirmation of successful gene editing events. Additionally, GFP does not confer antibiotic resistance, so there is no risk of conferring antibiotic resistance to other organisms.
**Transformation Protocol:** Protocol for CRISPR/Cas-9 gene editing using AAV viral vector for modifying immune genes, microbiota, and feather production genes in pigeons:
1. Adeno-associated virus vector (AAV) containing the desired genetic modifications and the fluorescent protein marker (e.g. GFP). 2. CRISPR/Cas9 gene editing plasmids. 3. Cell culture media (Eagle's MEM, Dulbecco's modified Eagle's medium (DMEM), Fetal bovine serum (FBS), penicillin and streptomycin). 4. Pipettes and pipette tips. 5. Mammalian cell lines 6. Surgical equipment for animal handling and DNA extraction.
1. Design and synthesize the CRISPR/Cas9 gene editing plasmids containing the desired modifications, using the promoters, enhancers and terminators selected for the specific gene editing. Verification of the design of the plasmids and its function is essential. 2. Package the CRISPR/Cas9 plasmids and the AAV vector inside a suitable viral delivery system suitable for transfection. 3. Propagate the viral vector in mammalian cell lines and culture cells in DMEM media supplemented with FBS (10% v/v) and penicillin/streptomycin (1%). 4. Check the viral titer and make sure they reach the required titre range for AAV-mediated transfection of target cells 5. Anesthetize the pigeons, and expose the site of injection for organ or tissue-specific delivery, (e.g., respiratory surfaces, digestive, or circulatory systems) with sterility precautions. 6. Deliver the viral vector, containing the desired gene editing tools into the tissues of the pigeon. The AAV vector can be delivered using methods such as injection or nebulization depending on the distribution mode of the viral vector. 7. Monitor the animals for any signs of illness or distress, and evaluate the efficiency and specificity of the gene editing by monitoring the fluorescence under microscopy and also molecular confirmation of the presence or absence of the genetic modification in the target tissue at several time points. 8. Analyze the tissues, and collect samples to confirm the success of the gene editing by extracting DNA from the modified tissues for molecular verification. 9. Repeat the process if further modifications or improvements are needed for the desired gene editing.
**Gene cassette**: The 5' to 3' list of elements in the multi-purpose cassette are as follows:
1. Ubiquitin C promoter 2. CRISPR/Cas-9 gene editing tool for targeting microbiota 3. Mitochondrial ATP Synthase Subunit 6 promoter 4. RNAi gene editing tool for targeting odor-causing genes 5. IgM gene enhancer 6. CRISPR/Cas-9 gene editing tool for targeting immune genes 7. Beta-actin promoter 8. Beta-globin enhancer 9. CRISPR/Cas-9 gene editing tool for targeting feather production genes 10. Beta-globin terminator 11. Bovine growth hormone terminator 12. Human alpha-globin gene terminator 13. GFP fluorescent protein marker
The cassette contains a combination of promoters, enhancers, CRISPR/Cas-9 gene editing tools, RNAi gene editing tools, terminators and a marker gene for selection. The cassette is designed for use in pigeons to modify their microbiota, immune genes, odor-producing genes, and feather production genes to make them sanitary. The cassette is assembled in a chosen vector, specifically the adeno-associated virus (AAV) vector, for efficient and targeted delivery of the gene editing tools for long-lasting effects in pigeons. The GFP fluorescent protein marker helps in the selection and identification of successfully modified organisms.
**Paper Abstract:** This project aims to develop genetic modifications in pigeons to make them sanitary by targeting the microbiota, immune system, odor-producing genes, and feather production genes. We plan to use gene editing techniques such as CRISPR/Cas-9 and RNA interference to achieve our goal. We will use a range of regulatory elements such as promoters, enhancers, and terminators to achieve optimal gene expression and transcription termination. To deliver the genetic modifications, we will use adeno-associated viral vectors due to their efficiency and long-lasting effect. If a selection marker is deemed necessary, we will use fluorescent protein markers such as GFP to select and identify successfully modified organisms. The successful implementation of these genetic modifications can result in a cleaner, healthier environment by reducing the spread of disease through improved pigeon hygiene.
**Growth, Selection & Stabilization:** Growth and Stabilization Protocol:
1. Growth Conditions: To grow healthy and disease-free pigeons, it is important to provide them with proper nutrition, clean water, and a clean environment. Pigeons should be fed a balanced diet consisting of pigeon feed and fresh fruits and vegetables. Their drinking water should be clean and free of contaminants.
2. Selection Conditions: For the CRISPR/Cas-9 gene editing of the microbiota, selection can be performed based on bacterial growth on selective media. Modified bacteria will have resistance to an antibiotic, which can be added to the media to select for modified bacteria.
For the RNAi knockdown of pathogenic genes, selection can be performed by monitoring the expression levels of the targeted gene using quantitative PCR or western blot analysis.
For the CRISPR/Cas-9 gene editing of immune genes, selection can be performed by monitoring the expression levels of the modified antimicrobial peptides using ELISA or western blot analysis.
For the RNAi knockdown of odor-causing genes, selection can be performed by monitoring the odor level of pigeon droppings or by analyzing the expression levels of genes involved in sulfur metabolism using quantitative PCR.
For the CRISPR/Cas-9 gene editing of feathers, selection can be performed by analyzing the morphology and composition of the feathers using microscopy and biochemical analysis.
3. Stabilization Protocol: To stabilize the modified organisms, it is important to maintain a stable microbiota and environment for the pigeons. Pigeons should be kept in clean and well-ventilated enclosures to prevent the growth of harmful bacteria and fungi. The microbiota can be stabilized by administering an appropriate dose of probiotics or prebiotics that promote the growth of beneficial bacteria.
Additionally, the modified pigeons should be monitored regularly to ensure that they are healthy and free of any adverse effects from the genetic modifications. Any observed negative effects should be addressed immediately to prevent further complications.
Overall, optimizing the growth and selection conditions and monitoring the stability of modified organisms can contribute significantly to developing a sustainable and safe approach to making pigeons sanitary.
**Proliferation Method:** To proliferate the final transformants once stabilized, we recommend selecting the modified cells using the fluorescent protein marker such as GFP, followed by propagation through breeding. The use of selection markers will aid in identifying and purifying cells that have successfully undergone gene modification. After selection, we recommend isolating cells that showed stable and reproducible expression of the modified genes. The cells can then be utilized to generate a breeding population.
We suggest using breeding as a means of proliferating the final population as it is a natural process that allows for the maintenance of the genetic modifications in the offspring. Pigeon populations can easily be maintained through breeding as they reproduce rapidly, and the transfer of modified genes in the offspring is more stable. This method ensures that the offspring will carry the desired genetic modifications, resulting in a stable and consistent population.
During breeding, it is important to ensure no inbreeding occurs as this can lead to a loss of genetic diversity and stability. Careful monitoring and selection of the breeding pairs can enable a preservation of genetic diversity while ensuring the genetic modifications are propagated. The use of markers such as fluorescent proteins or genotypic markers can aid in monitoring of genetic diversity.
In summary, to proliferate the final population, we recommend isolating stable and reproducible modified cells using selection markers, followed by propagation through breeding. This method allows for the maintenance of the desired genetic modifications in the offspring population, ensuring a stable and consistent population.
**Conclusion:** In conclusion, this project aimed to develop a genetic modification protocol for pigeons to make them sanitary, targeting microbiota, immune genes, odor-producing genes, and feather production genes. The use of CRISPR/Cas-9 gene editing tools and RNA interference provided promising results with the help of selection markers, promoters, enhancers, and terminators. The adeno-associated viral vector was found to be efficient and long-lasting for delivering the desired genetic modifications. Proper growth, selection, stabilization, and proliferation methods were also suggested to ensure the stability and safety of the modified organisms. The optimized gene construct developed in this project could be used in future studies for sustainable and safe pigeon breeding programs, leading to healthier populations of these essential urban wildlife.