1. Introduction to Animal Biotechnology:
Production of pharmaceutical proteins with
biotechnological methods. Transgenic technologies in animal organisms. The laboratory
mouse as a model organism. Biology, genetics and genome features of the laboratory
mouse. Oogenesis, spermatogenesis and fertilization in mammals. Critical embryo stages
in Animal Biotechnology. Bioethical issues.
2. Technologies for the generation of transgenic animals:
Functional Genomics
Technologies. Forward and Reverse Genetics. Creation of transgenic animals through
DNA microinjection into the pronuclei of fertilized oocytes. Creating transgenic animals
with lentiviruses. Advantages, Disadvantages. Transgenic animal applications.
3. Gene targeting technology:
Embryonic stem cells. Design of gene construct. Positive and
negative selection markers. Gene targeting stages for the generation of knockout mice.
Applications.
4. Genome modifications of animals with zinc-finger nucleases (ZFNs):
ZFNs features and
mechanism of action. Target Recognition by ZFNs. Design and construction of ZFNs.
Mechanisms of genomic modifications with ZFNs. Use in animal organisms and
applications.
5. Genome modifications of animals with the system CRISPR/CAS9:
The CRISPR/CAS gene
locus in the streptococcus. Description of the CRISPR/CAS9 system. Biotechnological use
of the CRISPR/CAS9 system in animal organisms. Strategies to minimize off-target
modifications. Comparison with ZNFs and TALENs nucleases.
6. Spatial and temporal control of inducible gene expression and modifications in
animals:
Recombination with the Cre/loxP system. Applications of the Cre/loxP system
to animal organisms. Tissue-specific gene activation and inactivation. Inducible temporal
gene modifications at the transcriptional and post-transcriptional level.
7. Forward Genetics:
from the phenotype to the identification of the causal gene:
Chemical mutagenesis with N-ethyl-N-nitrosourea. Phenotypic analysis. Detection of the
mutated gene by mapping. Stages of mapping. Polymorphic genetic markers.
8. Animal cloning:
Animal cloning methods. Nuclear transfer. Cloning stages. Reprogramming in cloning. Applications of animal cloning in agriculture and biotechnology.
Bioethical issues
9. Stem cells:
Types of mammalian stem cells and their topology. Embryonic stem cells.
Adult stem cells. Induced pluripotent stem cells. Stem cell applications.
10.Gene therapy:
History of gene therapy. Disease targets. Prerequisites and tools in gene
therapy. Ex vivo, in vivo gene therapy applications. Vectors in gene therapy. Stages of
gene therapy. Problems in gene therapy. Latest achievements.
11.Monoclonal antibodies – Vaccines:
Review of the immune system. Monoclonal,
polyclonal antibodies. Techniques for the production of monoclonal antibodies.
Applications of monoclonal antibodies in the diagnosis and treatment of human
diseases. Types of vaccines. Vaccination mechanisms. Examples, HPV vaccines.
Laboratory:
Generation of transgenic mice - Design of transgene construct - Isolation of
genomic DNA from mouse tissue - Genotyping of transgenic mice - Animal cell culture -
Overproduction of proteins in mammalian cell lines - Cryopreservation - In vitro fertilization (IVF), and intracytoplasmic sperm injection (ICSI) - Design of gene inactivation with the
CRISPR / CAS9 system.
• Molecular Biotechnology: Principles and Applications of Recombinant DNA. Glick BR,
Pasternak JJ, Patten CL. 4th edition, published by ASM Press. 2009.
• Principles of Gene Manipulation and Genomics. Twyman R, Primrose SB. 8
th edition, WileyBlackwell , 2013.
