Podcast for this lecture: https://notebooklm.google.com/notebook/18fcc724-1827-4694-ae0e-93a6f89b960c/audio
Epigenetics involves changes in gene expression that do not alter the DNA sequence itself but are instead influenced by chemical modifications and environmental factors. Epigenetics are the bridge between genotpye and phenotype. These modifications, which regulate gene expression, are heritable but reversible.
Epigenetics depend on the structural organization of DNA:
DNA needs to be accessible to transcription factors, machinery, RNA polymerase and enhancers/repressors. This depends on how tightly and loosely chromatin is bound.
How this works: Nucleosome protein complexes wind up DNA, these "beads on strings" can either be tightly packaged (not transcribable) or loosely packaged (transcribable)
The key mechanisms of epigenetic regulation include:
DNA Methylation:
All eukaryotic cells (except yeast)
Addition of a methyl group to cytosine residues in DNA, commonly in CpG islands found in gene promoter regions.
Suppresses gene expression by preventing transcription factor binding. Heritable during cell division, and can be denovo or to maintain methylation state (methylation is not permanent it is a dynamic process).
Plays a role in diseases like cancer:
Proto-oncogenes (growth factors): Hypomethylation (not enough methylation) = increased proliferation
Tumor surpressor genes (pro cell death): Hypermethylation of genes, reduced expression of regulating gene = increased proliferation
Histone Modification:
Modifications of histone proteins that either condense or relax chromatin structure, influencing gene accessibility for transcription. Possible action mechanisms:
Increase/decrease binding of effector molecules (TC factors, chromatin remodelling complexes)
Activate/repress gene transcription
Different types of modifications can either activate or repress gene expression.
Acetylation and Phosphorylation: Adds negative charge to histone = less attraction of DNA to histones = looser hold and greater translation
Ubiquination: can regulate DNA transcription by marking transcription factors or histones for degradation, thus suppressing transcription, or by modifying these proteins to enhance their activity, leading to transcriptional activation
Methylation: can activate or suppress DNA transcription by adding methyl groups to specific lysine or arginine residues on histones, which either condenses chromatin to repress transcription or relaxes it to enhance transcription, depending on the specific site and context of the modification.
RNA Interference (RNAi):
siRNA are specific for an mRNA transcript, bidning to their targets induce mRNA cleavage in the genome.
miRNA's target multiple mRNA regions and result in either cleavage and degredation of translational repression for a period of time.
Example: miRNA-152 regulates DNA methylation in the development and lactation process in cow mammary glands.
Binding leads to gobal DNA methylation leading to increase lactation related genes.
X-Chromosome Inactivation: Random inactivation of one X chromosome in female mammals prevents double expression of X-linked genes by tightly packing chomosome (main mechanism is high levels of DNA methylation)
Fun fact: genes for orange/black coat color are encoded on X-Chromosomes, so you can only observe calico colors in cats with two copies of X-chromosomes that both contibute to patterning.
Genomic Imprinting: Certain genes are expressed only from one parental allele, depending on methylation patterns inherited from the mother or father (mono-allelig gene expression). An example is growth regulation in liger and tigon hybrids due to differences in breeding strategies and hence imprints on growth genes.
External factors like nutrition can impact the epigenome. For instance, the Agouti viable yellow (Avy) mouse model demonstrates how dietary changes like genistein supplementation (donor of methylgroups) can alter DNA methylation and influence traits like obesity and coat color.
Supplemented: Increased DNA methylation on Avy allele = less expression = lower frequency of yellow mice and protection from obesity
Unsupplemented: Decreased DNA methylation on Avy allele = more expression = higher frequency of yellow mice and greater occurance of obesity.
Cancer:
TFIP-2 regulates MMP's that can cause cancer when over activated. Hypermethylation of tumor suppressor gene (TFPI-2 gene) in dogs promotes tumor growth = large B-cell lymphoma.
Tasmanian Devil Facial Tumor Disease: Epigenetic changes, such as reduced histone acetylation, results in lack of function of major histocompatibility complex (MHC) that prevents tumor cells from being recognized by the immune system.
Epigenome-wide association studies
Can show you the DNA methylation of the whole genome that can be correlated to certain conditions.
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1. Overview of Recombinant DNA Technology (rDNA)
Recombinant DNA technology is a series of methods to combine DNA segments from different sources to introduce desirable traits in organisms or produce specific proteins.
This technique involves creating recombinant DNA (rDNA) molecules, using vectors to insert DNA into host cells.
2. Applications
Medical: Therapeutic agents (e.g., insulin, cytokines), human proteins, vaccines.
Non-Medical: Biotechnology crops, genetic modifications for agricultural resilience.
Examples in veterinary medicine include protein therapies and gene therapies.
3. Vectors and Hosts for rDNA
Types of vectors include plasmids, cosmids, artificial chromosomes, and viral vectors.
Plasmids are small, circular DNA molecules that act as molecular vectors in recombinant DNA technology, with several key features that make them ideal for this purpose:
Replication Origin: Plasmids contain an origin of replication, allowing them to replicate independently within a host cell, ensuring the copied plasmid carries the foreign DNA.
Multiple Cloning Site (MCS): short DNA sequence found within plasmids and other vectors that contains several unique restriction enzyme (“molecular scissors”) recognition sites in a small, concentrated region. These restriction sites allow researchers to easily insert foreign DNA at a precise location in the plasmid.
Antibiotic Resistance Genes: These genes allow for easy selection of successfully transformed cells; only cells with the plasmid will survive in the presence of the antibiotic.
High Copy Number: Many plasmids are engineered to produce a high copy number in cells, yielding more recombinant DNA and higher protein expression when used in protein production.
Types of plasmids:
Cloning Vectors: These plasmids are primarily designed to carry and replicate a specific DNA fragment within a host. They have a multiple cloning site (MCS), an origin of replication, and often an antibiotic resistance gene for selection. Cloning vectors focus on amplifying or storing the inserted DNA rather than expressing it, making them ideal for DNA manipulation, sequencing, or transfer to another vector.
Expression Vectors: Expression vectors are engineered to not only carry foreign DNA but also to enable its transcription and translation within the host cell, resulting in protein production. They contain all cloning vector features plus additional elements, such as a promoter for gene expression, regulatory sequences for controlled expression, and sometimes a tag for protein purification. These vectors are ideal for producing recombinant proteins in research or therapeutics.
4. Enzymes used in recombinant DNA technology:
Restriction Enzymes: These are proteins that act as molecular "scissors," cutting DNA at specific sequences, known as restriction sites. Each enzyme recognizes a particular sequence (often palindromic) and makes precise cuts. Some produce "sticky ends" with single-stranded overhangs, while others make "blunt ends." Sticky ends can pair with complementary sticky ends, making it easier to join DNA fragments.
DNA Ligases: DNA ligases act as "molecular glue," sealing breaks in the DNA backbone by forming phosphodiester bonds. After restriction enzymes cut both the plasmid and foreign DNA to create compatible ends, DNA ligase joins these fragments, stabilizing the new recombinant DNA. This step is crucial for creating continuous, functional DNA molecules in cloning and genetic engineering.
5. Tools and Procedures in Recombinant DNA
Obtain the Target Gene (Sequencing and PCR):
First, researchers identify and isolate the gene of interest that encodes the desired protein. This often involves sequencing the DNA to confirm the exact genetic code.
Polymerase Chain Reaction (PCR) is used to amplify (make multiple copies of) this gene from a DNA sample, creating enough of the target DNA for cloning. PCR enables precise isolation and amplification of the gene, allowing it to be inserted into a vector.
Insert the Gene into a Vector to Create Recombinant DNA:
A vector, commonly a plasmid, is prepared to carry the target gene. Using restriction enzymes, specific sequences are cut in both the vector and the target gene to create compatible ends. These enzymes make either “sticky” or “blunt” ends on the DNA, allowing the foreign gene to insert into the vector.
DNA ligase is then added to “glue” the ends together, forming a stable recombinant DNA (rDNA) molecule. This recombinant plasmid now contains the target gene and can be used to introduce it into host cells.
Transform rDNA into Bacterial Cells (e.g., E. coli):
The recombinant plasmid is introduced into bacterial cells, such as E. coli, through a process called transformation. This step enables the host bacteria to take up and replicate the recombinant plasmid.
Transformation techniques include chemical methods (using CaCl₂) or electroporation, which briefly shocks cells to make their membranes more permeable to DNA.
Select and Confirm rDNA via PCR or Sequencing:
After transformation, only some bacterial cells will contain the recombinant plasmid. To identify these, an antibiotic resistance gene on the plasmid allows researchers to grow only the transformed bacteria on antibiotic-containing media.
PCR or sequencing is used to confirm that the bacteria contain the correct recombinant plasmid with the target gene. This ensures the accuracy of the cloning process.
Transfer rDNA to Host Cells (Prokaryotic or Eukaryotic):
The recombinant DNA is then transferred to a “protein production factory” or host cell. For simpler proteins, E. coli or other bacterial cells are common choices. For complex proteins needing post-translational modifications, eukaryotic cells like yeast or mammalian cells are preferred.
Depending on the host, different vectors (plasmids for bacteria, viral vectors for eukaryotes) and transformation techniques are used.
Confirm Expression with SDS-PAGE:
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) is used to confirm the production of the target protein. In SDS-PAGE, proteins are separated by size on a gel, allowing researchers to verify the presence and approximate size of the expressed protein.
This method provides visual evidence that the host cells are producing the recombinant protein as expected.
Purify and Scale Up Protein Production:
The protein is then purified from other cellular components using techniques like affinity chromatography, which binds and isolates the target protein based on unique properties (e.g., affinity tags like His-tag).
For larger quantities, the process is scaled up by culturing the host cells in large bioreactors or fermenters. This step is crucial for applications requiring substantial amounts of the protein, such as therapeutics and industrial enzymes.
6. Host cell selection:
Host Cells are selected based on the requirements for protein production:
E. coli: E. coli is a common prokaryotic host in recombinant DNA technology due to its rapid growth rate, ease of cultivation, and high protein yield, making it cost-effective for producing simpler proteins. However, as a prokaryote, E. coli lacks the machinery for post-translational modifications (e.g., glycosylation, phosphorylation) that are often necessary for the functionality of complex proteins, especially those used in therapeutic applications.
Mammalian Cells: Mammalian cells, such as Chinese hamster ovary (CHO) cells, are eukaryotic and provide the full range of post-translational modifications needed for complex protein folding and functionalization. These cells more closely replicate human cellular environments, making them ideal for producing complex therapeutic proteins. Though they are slower-growing and more costly to maintain, mammalian cells are essential for proteins requiring a structure and function similar to that in humans, such as antibodies and hormones.
7. Insulin as a Case Study in Protein Engineering
First-Generation Recombinant Insulin
Initially, first-generation insulin was produced by cloning the separate A and B chains of human insulin into E. coli, which then expressed each chain separately. These chains were purified and chemically combined in vitro to form disulfide bonds, resulting in active insulin. This method allowed large-scale production, replacing animal-sourced insulin. However, the in vitro formation of disulfide bonds required precise conditions and was challenging to scale due to:
Insulin Structure Complexity: Insulin is produced as a precursor, pro-insulin, that requires specific disulfide bonds to form between the A and B chains after removal of the connecting C chain. This process does not naturally occur in E. coli, which complicates production.
Limitations in Pharmacokinetics: First-generation insulin did not fully mimic the rapid absorption and release rates of natural insulin in humans, causing slower onset and extended duration of action, which could lead to delayed or prolonged effects in patients.
Second-Generation Insulin: Enhanced Pharmacokinetics
Advances in second-generation insulins sought to address these pharmacokinetic limitations by engineering insulin analogs with modified absorption and release characteristics to better match the body's needs.
Insulin Lispro (Fast-Acting):
Lispro was engineered by swapping two amino acids (proline and lysine) in the B chain, which inhibits dimerization and hexamer formation (insulin tends to form stable complexes that slow its release).
This substitution prevents molecules from binding tightly, allowing faster diffusion into the bloodstream after injection.
Lispro provides rapid action, with onset within 15 minutes, a peak at 30–90 minutes, and a shorter duration (around 5 hours), making it suitable for quick blood glucose control around meals.
Insulin Glargine (Long-Acting):
Glargine was modified to provide a steady, prolonged release by changing its isoelectric point. Two arginine residues were added to the B chain, and the A chain’s terminal asparagine was replaced with glycine.
These modifications make Glargine more soluble at acidic pH and less soluble at the body’s neutral pH. Upon injection, it forms microprecipitates under the skin that slowly dissolve, leading to a steady insulin release over 24 hours.
This reduces the need for frequent injections, providing stable, baseline insulin levels.
Key Improvements and Benefits of Second-Generation Insulins
Enhanced Control: Lispro and Glargine provide tailored insulin actions that allow for precise blood sugar control, mimicking natural insulin more closely.
Reduced Injection Frequency: Long-acting Glargine reduces the number of daily injections required.
Reduced Risk of Hypoglycemia: Better control over insulin release helps minimize the risk of sudden blood sugar drops.
8. Therapeutic Antibodies: Overview
Therapeutic antibodies, particularly immunoglobulins (e.g., IgG), are engineered proteins that bind to specific antigens with high specificity. By recognizing and neutralizing foreign substances, they have applications in treating allergies, autoimmune diseases, cancers, and infections.
Monoclonal Antibodies Production: Monoclonal antibodies are produced through the fusion of antibody-producing B-cells (from an immunized animal) with myeloma cells, creating a hybridoma cell line. This process provides several benefits:
Immortalization: Myeloma cells allow the hybridomas to proliferate indefinitely in culture.
Specificity and Consistency: Each hybridoma cell line produces a single, specific antibody consistently, ensuring high specificity in treatment.
Caninization for Veterinary Use: For veterinary applications, antibodies often require species-specific modifications to avoid immune responses. To make antibodies more suitable for dogs, researchers use caninization, a technique to modify antibodies derived from one species (like mice) to make them compatible with the canine immune system. This is done by combining the variable regions of the antibody (from the murine antibody) with constant regions from canine IgG. This minimizes immune reactions and improves the antibody’s effectiveness and longevity in dogs.
Lokivetmab:a monoclonal antibody specifically designed for treating atopic dermatitis in dogs. Atopic dermatitis is an allergic, inflammatory skin condition causing severe itching. Lokivetmab works by targeting interleukin-31 (IL-31), a signaling molecule involved in itch sensation. By binding to IL-31, Lokivetmab prevents it from activating receptors on nerve cells in the skin, effectively reducing itchiness.
Key Features of Lokivetmab:
Species-Specific Design: Lokivetmab is "caninized," making it less likely to provoke an immune response in dogs.
Rapid Relief and Long Duration: Lokivetmab can provide relief within a day of injection, with effects lasting 4–8 weeks.
Targeted Action: Its specific action on IL-31 makes it a targeted therapy with fewer side effects compared to general immunosuppressants, such as corticosteroids.
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1. Animal Cloning
Cloning Definition: The process of creating genetically identical copies of cells, tissues, or entire organisms.
Natural Clones: Identical twins occur through natural processes.
Artificial Cloning Methods:
Embryo Twinning: Separating cells at early cell division after fertilization (blastomeres) at early embryonic stages to mimic natural twin formation. Each blastomere can form an individual organism.
Somatic Cell Nuclear Transfer (SCNT): Replacing an oocyte’s nucleus with the nucleus from a somatic cell (ex: fibroblasts), allowing reprogramming to a pluripotent state. Blastocyst develops into a fetus in a surrogate and creates a clone of the nucleus donor. This technique produced Dolly the sheep, the first cloned mammal (late 1990’s).
This technology allowed us to establish the technology to reprogram a differentiated cell to an induced pluripotent stem cell.
Applications: Conservation efforts to preserve species, pet cloning, and livestock with desirable traits.
Example: Using dog oocytes to clone coyotes. Sounds great, but was problematic because the mitochondrial DNA from mom was not compatible, and the surrogate mother’s immune system recognized the embryo as foreign.
Challenges: Reprogramming errors can lead to pregnancy loss, developmental issues, congenital abnormalities, and early postnatal death.
2. Genetically Modified Organisms (GMOs) & Transgenic Animals
Definition: Organisms altered through genetic engineering, allowing gene deletion, insertion, or modification.
Applications:
Enhanced animal products or health (e.g., disease resistance).
Creation of disease models for research.
Bioreactors for biomedically relevant proteins (e.g., goats producing spider silk proteins).
Techniques:
Somatic Cell Nuclear Transfer (SCNT) with transgenic cells involves creating genetically modified animals by introducing specific DNA changes into cultured somatic cells before using these cells to produce clones.
Creation of Transgenic Cells: Somatic cells (such as fibroblasts) are cultured and manipulated in the lab to introduce desired transgenes (foreign DNA) into their genomes. This DNA can integrate randomly into the cell's genome, potentially resulting in expression changes.
Nuclear Transfer: The nucleus of a transgenic somatic cell is then transferred into an enucleated oocyte (an egg cell with its nucleus removed). This oocyte now contains the transgenic nucleus, which includes the modified DNA.
Embryo Development and Transfer: The modified oocyte begins to develop into an embryo, which is then implanted into a surrogate mother. The resulting offspring will be a genetic clone of the transgenic somatic cell donor, with the introduced gene modifications
Challenges: transgenes integrate randomly in the genome (no control over where/how many copies introduced) which may lead to gene silencing or overexpression (can be toxic)
3. Genome Editing
Overview: The ability to precisely edit DNA at specific sites, utilizing cellular DNA repair mechanisms in response to double-strand breaks (DSBs). Requires a nucleus and mechanism to direct the DNA to a specific location in the genome.
Applications:
Animals as bioreactors
Biomedicine/organs
Double stranded break (DSB) can be accomplished through:
Endogenous processes (DNA replication machinery)
Exogenous agents (UV radiants, chemicals)
Repair Mechanisms:
Non-Homologous End Joining (NHEJ): repairs DNA by directly ligating the ends of double-strand breaks without using a homologous template, which can lead to errors like insertions or deletions at the repair site.
Error-prone, often introducing indels, leading to frameshifts/gene knock-outs
Homology-Directed Repair (HDR): repairs DNA double-strand breaks by using a homologous DNA template to accurately guide the repair, allowing precise insertion, deletion, or replacement of DNA sequences.
Genome Editing Tools:
Meganucleases: Endonucleases with long DNA recognition sequences (12–40 base pairs) that enable highly specific DNA cleavage. Due to the specificity of their binding sites, they have fewer off-target effects but are not feasible for regular use because their highly target-specific capacity limits their flexibility, making it challenging to re-engineer for new target sites.
Zinc Finger Nucleases (ZFNs): Custom-designed nucleases composed of zinc finger domains that recognize specific DNA triplets, coupled with the FokI nuclease domain, which cleaves DNA only when two ZFN proteins bind adjacent DNA sequences. This dimerization requirement enhances specificity but makes engineering new targets complex and time-consuming.
TALENs (Transcription Activator-Like Effector Nucleases): Use transcription activator-like effector (TALE) proteins to bind specific DNA sequences, recognizing each nucleotide individually with a repeat-variable diresidue (RVD) code. Paired with FokI nuclease domains, TALENs induce a double-strand break upon dimerization, offering high specificity and easier customization than ZFNs.
CRISPR/Cas9: Uses a guide RNA (gRNA) complementary to a 17-20 bp DNA sequence to direct the Cas9 nuclease to the target site, where it introduces a double-strand break. The simplicity of gRNA design makes CRISPR/Cas9 highly adaptable for new targets, although it carries a higher risk of off-target effects compared to protein-based recognition systems.
4. Examples of Genome Editing
Hornless Cows: Using TALENs to insert genes for hornlessness into Holstein cattle, aiming to improve animal welfare by avoiding painful dehorning practices.
Target Site Identification: Researchers identified a specific genetic variant on chromosome 1 found in naturally hornless cattle breeds, which involves a 10 bp deletion and a 212 bp insertion that makes these breeds hornless.
Designing TALENs: TALENs were custom-designed to recognize and bind to the DNA sequence flanking the target site in the Holstein cattle genome. Each TALEN protein consists of DNA-binding TALE domains that recognize specific nucleotides and a FokI nuclease domain that introduces a double-strand break when two TALENs bind to adjacent DNA sequences.
Double-Strand Break and Donor Template: After TALENs bind to the target site, the FokI nucleases create a double-strand break. A repair template containing the hornless variant sequence (10 bp deletion and 212 bp insertion) is also provided. This template has homologous sequences that match the cut ends of the DNA, allowing it to serve as a guide for repair.
Homology-Directed Repair (HDR): The cell’s homology-directed repair mechanism uses the provided template to repair the double-strand break, incorporating the hornless genetic variant sequence into the Holstein genome at the target site.
Embryo Transfer and Breeding: Once edited, the modified embryos are implanted into surrogate cows. The calves born from these embryos inherit the hornless trait through the introduced variant.
PRRSV-Resistant Pigs: CRISPR/Cas9 was used to create pigs resistant to Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).
Target Selection: Researchers identified Exon 7 of the CD163 gene, which contains a segment crucial for the virus’s entry into pig cells. This exon includes the scavenger receptor cysteine-rich domain 5 (SRCR5), the site PRRSV binds to.
CRISPR Editing: Using CRISPR/Cas9, guide RNAs were designed to specifically target and cut DNA in Exon 7 of CD163 in pig zygotes. The Cas9 enzyme, directed by the guide RNAs, made a precise double-strand break at the target site.
Gene Deletion: After the DNA break, the cell’s repair mechanism (often via non-homologous end joining) led to a deletion of the Exon 7 region, effectively removing SRCR5. This modification disrupted the virus’s ability to recognize and bind to the CD163 receptor.
Embryo Transfer and Development: The edited zygotes were implanted into surrogate sows, which gave birth to piglets with the modified CD163 gene.
Result: These genetically modified pigs lacked the SRCR5 domain but retained other functions of the CD163 protein, making them resistant to PRRSV infection. Tests showed that these pigs did not develop PRRSV symptoms, as the virus could no longer use CD163 to infect their cells.
Human Applications: Gene-editing to treat genetic disorders:
Stem Cell Extraction: Hematopoietic stem cells, responsible for producing blood cells, are collected from the patient’s bone marrow or blood.
CRISPR Editing: CRISPR/Cas9 is applied to these stem cells to knock out or disrupt the BCL11A gene. By disabling BCL11A, the suppression of fetal hemoglobin production is lifted, allowing the cells to produce HbF instead of the defective adult hemoglobin (HbA).
Stem Cell Infusion: After gene editing, the modified stem cells are expanded and infused back into the patient. The patient undergoes conditioning (e.g., chemotherapy) to eliminate existing bone marrow cells, making space for the gene-edited cells.
Production of Fetal Hemoglobin: The edited stem cells engraft in the patient’s bone marrow, continuously producing red blood cells with functional fetal hemoglobin. HbF can effectively carry oxygen and is not affected by the mutations causing sickle cell anemia or beta-thalassemia, thus alleviating symptoms and reducing complications.