TEMA 3.1 Y 3.2 - ANIMAL TRANSGENESIS

SECTION I: From Genes to Cells in Biotechnology

Lesson 1: Gene Cloning and Recombinant Protein Expression

1.1 Cloning (Last Generation Approaches)

  • Definition of cloning: The process of creating an identical copy of an organism or of a  specific DNA sequences (molecular cloning)

  • Last generation cloning approaches include methods such as CRISPR and other advanced molecular techniques that allow for precise editing and modification of genetic material.

1.2 System for Recombinant Protein Expression

  • Recombinant protein expression: the process of producing proteins from recombinant DNA, which is formed by combining DNA from different sources (like a human gene inserted into a bacterial plasmid)

  • Involves the introduction of coding sequences into expression vectors that are then transfected into host cells, leading to expression and production of the desired protein.

  • ON MAMMALIAN HOST CELLS: best for complex glycosylation and folding, high fidelity to native human proteins, slower growth, higher cost.

  • EXPRESSION VECTORS: specialized plasmids or viruses that drive high levels of gene transcription and translation into a host cell. Contain:

    • STRONG PROMOTER to initiate robust transcription

    • RBS or kozak sequence: ribosomal binding site to facilitate efficient translation from mRNA to protein

    • terminator sequence: signals end of transcription

    • ORI (origin of replication) for vector maintenance and amplification.

    • selector gene / selectable marker gene: allows ID of host cells that have successfully taken up the recombinant vector.

Lesson 2: Gene Transfer Technologies into Eukaryote Cells

2.1 Non-viral Technologies of Gene Transfer

  • Overview:

    • non-viral methods of gene transfer can be physical or chemical - directly deliver genetic material INTO EUKARYOTIC CELLS.

    • They have lower immunogenicity and larger cargo capacity compared to viral methods - can carry larger molecules.

      • PHYSICAL: techniques such as

        • electroporation (fuck with the membrane, open pores that molecules can pass through via electric shock) 

        • microinjection (direct injection of DNA into the nucleus or cytoplasm of a single cell using a fine glass needle. PRECISE DELIVERY TO INDIVIDUAL CELL - labor intensive and low throughput) 

        • lipofection (LIPID MEDIATED GENE TRANSFER- DNA IS ENCAPSULATED with lipid vesicles called liposomes that fuse with the cell membrane, delivering genetic material into the cell.

2.2 Viral Technology of Gene Transfer

  • Vectors: Utilizes modified viruses to deliver genetic material into cells.

  • Types of viral vectors include adenoviruses, lentiviruses, and retroviruses, each with unique advantages and disadvantages in terms of integration into host genomes.

  • THEY ARE REPLICATION INCOMPETENT - PATHOGENIC VIRAL GENES ARE REMOVED AND REPLACED WITH THE THERAPEUTIC GENE MAKING THEM UNABLE TO CAUSE INFECTION. THEY ARE SAFE.

    • RNA VIRUSES:  LENTIVIRUS (CAN INFECT BOTH DIVIDING AND NON DIVIDING CELLS AND INTEGRATE INTO HOST GENOME), RETROVIRUS (INFECT ONLY DIVIDING CELLS, INTEGRATE INTO HOST GENOME - STRICT REQUIREMENT FOR CELL DIVISION LIMITS THEIR APPLICATION)

    • dsDNA VIRUSES:ADENOVIRUS (EPISOMAL, DOES NOT INTEGRATE INTO HOST CHROMOSOMES) CAN CARRY LARGE DNA INSERTS FOR TRANSIENT EXPRESSION, HERPES VIRUS

    • ssDNA VIRUS: PARVOVIRUS, ADENO-ASSOCIATED VIRUSES (CAN INFECT DIVIDING AND NON-DIVIDING CELLS, EPISOMAL, INTEGRATE AT A SPECIFIC CHROMOSOME, CONSIDERED SAFE IN GENE THERAPY, SMALL, LIMITED CARGO CAPACITY)

Seminar: Mass Sequencing (Albert Ferrer)

  • Discussion on advancements in sequencing technologies, their applications, and implications for biotechnology and genomics.

    • SANGER SEQUENCING: 1st generation, method based on chain termination. accurate reads, low throughput.

    • NEXT-GEN SEQUENCING: 2nd generation, parallel sequencing of millions of shorter DNA fragments at the same time, enabling higher throughput and reduced costs, which has revolutionized genomics and allowed for the comprehensive analysis of complex genomes. whole genome sequencing, RNA-seq, ChIP-seq.

    • THIRD GENERATION SEQUENCING: long-read sequencing technologies that read single-strand DNA directly. PacBio and Oxford Nanopore - more complex applications like repetitive regions, structural variants, and de novo assembly. for now, higher error rates than NGS.

SECTION II: From Cells to Organisms: Transgenesis in Biotechnology

Lesson 3: Transgenesis

3.1 Animal Transgenesis (Introduction with Evo-Devo Perspective) (Cristian Cañestro)

3.1.1 Methodologies to Generate Transgenic Animals

  • Transgenesis: stable introduction of exogenous genetic material (this is a transgene) into the germline of an animal, such that it is transmitted to subsequent generations via reproduction. RESULTS IN A TRANSGENIC ORGANISM THAT EXPRESSES A NEW TRAIT.

  • Techniques for generating transgenic animals include:

    • microinjection into zygotes: direct microinjection of DNA into the pronucleus of a fertilized egg. ZYGOTE. FEM ZYGOTE. the whole point is that the transgene integrates into the host genome BEFORE the first division. WHY DO I WANT THIS: ONLY WAY TO GUARANTEE THAT THE TRANSGENE IS INCORPORATED INTO THE GERMLINE, ALLOWING YOU TO ESTABLISH A PERMANENT, HERITABLE TRANSGENIC LINE. If integration happens later, you risk creating a mosaic animal that cannot reliably pass the gene to the next generation. efficiency varies with random integration patterns.

    • somatic cell nuclear transfer (SCNT): Involves transferring the nucleus from a somatic cell (containing the desired transgenic modification) into an enucleated oocyte. This reconstructed egg is then activated to develop into an embryo. allows for precise manipulation of the somatic cell before cloning, enabling the creation of genetically identical transgenic animals with defined genetic changes.

    • the use of embryonic stem cells (ESCs): ESCs are pluripotent stem cells derived from the inner mass of a blastocyst.

      • can be genetically modified in vitro - homologous recombination for targeted gene knock-outs or knock-ins, and then injected into a host blastocyst. The resulting chimera can produce offspring with the modified germline if the ESCs contribute to the germline, leading to stable transmission of the transgene. high targeting efficiency for specific gene modifications.

  • Each method varies in efficiency, integration patterns, and ethical considerations.

  • WHEN TO PICK WHAT METHOD:

    • so technically you could use CRISPR/Cas9 for precise gene editing and site-directed mutagenesis in any of them. the delivery method affects what you’re delivering into the initial cell - like if you want a stable transgenic line you should do it as early as you can, hence zygote microinjection. what changes is that the genetic material being introduced has been engineered for site directed mutagenesis.

    • If this targeted modification occurs early enough to be present in the germline of the resulting animal, it will lead to a stable transgenic line that reliably passes the engineered transgene or specific mutation to subsequent generations.

    • traditional zygote microinjection, especially for introducing larger transgenes without specific targeting tools, often results in random integration into the genome,

    • its application has significantly evolved with modern gene editing technologies. Zygote microinjection is a primary method for delivering the components for site-directed mutagenesis (e.g., CRISPR-Cas9 system reagents like Cas9 mRNA and guide RNAs) directly into the fertilized egg's pronucleus or cytoplasm.

    • So, while the delivery method is microinjection, the mechanism of the mutation is the highly precise gene-editing tool.

    • This contrasts with SCNT, which allows for the thorough validation of complex, targeted edits in somatic cells before cloning, ensuring precise outcomes prior to animal generation and offering advantages for very complex or multi-locus modifications where pre-screening is beneficial.

    • SCNT allows for the engineered mutations to be introduced to somatic cell and VERIFIED/ validated before animal is even cloned: This pre-screening and validation step is a major benefit because it ensures a precise outcome. By confirming the edits before generating an animal, scientists can avoid the uncertainty and potential inefficiencies of methods where modifications are introduced directly into an early embryo. use in highly complex scenarios, doll.

    • si te preguntan por quien puede ser random, es zygote injection la que se usaba para random insertion (variable efficiency).

3.1.2 Experimental Transgenic Approaches

  • experimental design: careful planning

    • selecting appropriate promoter to drive transgene expression

      • tissue specific, inducible, constitutive (ubiquitous, STRONG)

    • designing expression cassette

      • gene of interest, regulatory elements, loxP sites, selector genes

    • choosing/developing animal model

    • establishing appropriate control groups

      • wild type, non transgenic siblings

  • validation methods: hay que confirmar la presencia and expression of the transgene.

    • Genomic DNA analysis:

      • PCR to detect the presence of the transgene

      • Southern blot to confirm gene copy number and integration pattern

    • RNA analysis:

      • Northern blot or RT-qPCR to verify transgene mRNA expression levels

    • Protein Analysis:

      • Western Blot, ELISA, immunohistochemistry to confirm transgene expression, localization, and expression levels across different tissues

    • Phenotypic Analysis: assess functional impact of the transgene by observing and measuring the observable characteristics (phenotype) of an organism and comparing them to appropriate control groups (like wild-type or non-transgenic siblings).

      • includes changes in morphology, histology, physiology, behavior, disease susceptibility or resistance, or any other measurable outcomes that indicate how the transgenic protein ias affecting the organism’s biological processes and general health.

  • ethical concerns related to transgenic research: animal welfare (suffer as little as possible), animal use, resource use, all the shit around whether using an embryo is ethical, biodiversity, dignity, suffering.

3.1.3 Applications of Animal Transgenesis

  • Applications include developing models for human diseases, agricultural improvements, and biopharmaceutical production.

    • agricultural improvements:

      • livestock productivity

      • meat quality, new qualities to animal products, enhanced resistance to diseases.

3.2 Plant Transgenesis (Teresa Lant)

  • Summary of methods for creating transgenic plants and their agricultural applications.

  • AGRICULTURAL APPLICATIONS OF TRANSGENIC PLANTS:

    • herbicide tolerance

    • insect resistance

    • disease resistance

    • improved nutritional value

    • stress tolerance

    • delayed ripening/improved shelf life

  • METHODS TO MAKE TRANSGENIC PLANTS:

    • Agrobacterium-mediated transformation: Utilizes the natural ability of Agrobacterium tumefaciens to transfer a portion of its DNA into the plant genome, effectively introducing new genes.

    • Particle bombardment via GUN! shoot new genes into cell wall of plant cells in gold or tungsten microscopic bullets. use for corn and rice and plants that resist agrobacterium.

    • protoplast transformation: involves removing the cell walls of plant cells to create protoplasts, which can then take up DNA directly via chemical (e.g., polyethylene glycol) or electrical (electroporation) methods

Seminar: Course Review by the Use of Oligonucleotides in Different Biotechnological Contexts (Ramon Eritja)

  • Provides a comprehensive overview of oligonucleotide applications including diagnostics, therapeutics, and synthetic biology.

OVERVIEW

Genetic and Molecular Bases of Biotechnology (GMBB)

  • Introduction to the foundational concepts and principles in biotechnology related to genetics and molecular biology.

Gene Information and Definitions

1. General Concepts of Animal Transgenesis

  • Genetically Modified Organism (GMO): "Organismo modificado genéticamente (GMO): cualquier organismo, cuyo material genético ha sido modificado de una manera que no se produce de forma natural en el apareamiento o en la recombinación natural."

  • Transgenic Organism: A specific subtype of GMO that has had foreign DNA (transgene) introduced into its genome. This allows for traits not naturally present in the species to be expressed.

  • ALL TRANSGENIC ANIMALS ARE GMOs but NOT ALL GMOs are TRANSGENIC ANIMALS. it’s a rectangle-square situation.

  • Genetically Edited Organism: A GMO whose DNA has been modified using precise methods, such as CRISPR, to achieve targeted genetic changes. Unlike classical mutagenesis, these edits are specific, minimizing unintended consequences.

Ethical and Regulatory Discussions

  • Discussion on EU legislation considering CRISPR plants as transgenics, counterarguments about regulation stifling innovation, and the importance of consumer rights in terms of labeling GMO products.

Transgenic Techniques

Zygote Microinjection

  • A technique involving the direct injection of DNA into the pronucleus of a fertilized egg, which allows the foreign DNA to integrate into the host genome during cell division.

Considerations for microinjection:

  1. Timing of Injection: Injection must occur at the right developmental stage to ensure integration. BEFORE THE FIRST DIVISION!!!!!

  2. Potential Efficiency: Different species and developmental stages influence success rates of the technique.

Retroviral Vectors

  • Used to introduce genetic material into cells where integration is required; retroviral vectors allow stable gene transfer and expression. RETROVIRUS HAS INTEGRATION. ADENOVIRUS DOES NOT. ADENOASSOCIATED TAMPOCO.

Embryonic Stem Cells and SCNT

  • ESCs can differentiate into any cell type and are manipulated to introduce desired genetic changes before being introduced back into the organism to produce transgenic offspring.

  • SCNT involves transferring nuclei from somatic cells into enucleated oocytes, producing cloned and potentially transgenic animals.

Applications and Future Prospects

  • Applications include creating models for studying human diseases, improving crop sustainability, and animal husbandry.