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Biotechnology Gene therapy, Cloning Heredity No 439 by RNDr. Hana Zoubková, PhD What is Biotechnology? The application of technology to improve a biological organism by changing or adding genes from another organism …………...produce Genetically modified organisms Gene engineering, transgenosis, transgenic organism What is Cloning? The process of producing populations of genetically identical individuals Molecular cloning – amplification of DNA fragments Cell cloning – an application of stem cells Organism cloning – somatic cell nuclear-transfer What is Gene therapy? The application of technology to improve (to heal) a human organism by changing or adding genes from another organism Cloning of cell or organism Reproductive vs. therapeutic cloning An aim of reproductive cloning is origin of a baby An aim of therapeutic cloning is to provide stem cells for a patient, which requires a transplant Technique of embryo division – old technique of formation genetically identical individuals, division of morula or blastocyst Technique of nucleus transfer a transfer of somatic cell nucleus to enucleated egg Stem cells Embryonic stem cells – an application is forbidden Infant and adult stem cells – an application is permitted and they are used for therapy. They are present in small numbers in Bone marrow Peripheral blood Skin epithelium Umbilical cord blood Dental pulp of infant’s teeth Stem cells may be obtained by reprogramming somatic cells Sources of adult and infant stem cells Gene therapy Somatic A genome is changed, but the change is not passed to other generation. The gene in patients cells are repaired and returned back. Germ gene therapy A genome is changed directly in ovum or sperm cell and the change is passed to other generations - is not proceeded for any kind of animals Researchers may use several approaches A transgen may be inserted into a non-specific location within the genome to replace a function of an abnormal gene. An abnormal gene might be swapped for a normal gene through homologous recombination. An abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function. An gene might be turned on or off, a regulation of its expression is altered. 1 2 4 3 Ex vivo – pathologic tissue is taken from patient. Cells accept transgen in laboratory conditions and after that are cells given back to patient In vivo – we prepare viral vectors with transgene and we introduce (infect) patient‘s tissue with the viral vectors Problems: Multifactorial disorders Short-lived nature of gene therapy Immune response The genes are not expressed in intended tissues and in intended place of the genome. Examples of gene therapy trials for inherited disorders Disorder Cells altered Gene therapy strategy SCID - ADA deficiency T cells and hemopoietic stem cells Ex vivo GAT using recombinant retroviruses containing an ADA gene Cystic fibrosis Respiratory epithelium Familial hypercholesterolemia Liver cells Gaucher's disease glucocerebrosidase Hemopoietic stem cells In vivo GAT using recombinant adenoviruses or liposomes to deliver the CFTR gene Ex vivo GAT using recombinant retroviruses to deliver the LDL receptor gene (LDLR) Ex vivo GAT using retroviruses to deliver the gene (GBA) Molecular cloning - steps Gene manipulation and introduction and multiplication 1. An identification and isolation of gene 2. We modify an existing gene (a new allele) or we create gene knock-out or we modify foreign DNA or synthetic genes to be transgene 3. The introduction of transgene into an organism for multiplication 4. Selection of transgenic organisms and a usage of multiplicated transgene or its protein Molecular cloning: the principle of bacterial production of insulin, it is used a vector with transgene (insulin gene) 1. Identification, isolating unknown gene by Homology cloning based on a similarity to known genes. e.g. Mouse‘s gene will help to determine the localization of human one in hybridization method Complementary genetics we predict nucleotide sequences due to known aminoacids sequences Map-based cloning based on searching of genetic markers in linkage with the unknown gene – chromosome walking 2. Preparation of recombinant DNA which contains polylinker: artificially synthetized part of DNA with specific spots for restriction endonucleases (RE), which allow to incorporate gene of interest transgen, also contains resistance to two or more antibiotics and origin of replication. The whole recombinant DNA must be short enough for vector. ● Gene of interest ● Selectable marker ● Insertion sequences Isolated gene (gene of interest) becomes transgene - the gene must be combined with other genetic elements in order to be expressed properly. The gene can also be modified at this stage for better expression or effectiveness. Promoter TP Coding Region Promoter Region and Terminator region Controls when, where and how much the gene is expressed - viral promotors Transit Peptide Targets protein to correct organelle Coding Region Encodes protein product vectors for transgenosis Bacterial plasmids Bacteriophage lambda (λ) Cosmids - combination phages and plasmids Integrated vectors Artificial yeast chromosome, YAC Viruses (adenoviruses, retroviruses, lentivirus) – mammals and humans Restriction endonuclease enzymes, which cut DNA molecules at specific recognition nucleotide sequences known as restriction sites palindroms mostly bacterial enzymes 3. Insertion of vector to genome: Transgenic mammals and gene therapy: introduction of viral vectors, retroviruses, adenoviruses, lentiviruses with recombinant DNA and transgene Plant and bacterial transgenosis: introduction of plasmids, phages, cosmids with recombinant DNA and transgene by electric, heat or sound impulse, mikroinjection, gene shooting, liposomes 4. selection of transformed organism and an application of multiplied transgene or its protein in a basic research of the gene and protein or a medical, food or agricultural sciences and industry. Transgenic plants with resistance and higher profitability. Transgenic bacteria able to clean toxic wastes. The application of protein generated by transgene for basic research and for clinical use: insulin, human growth hormon … Application in Medicine ….also human albumin, monoclonal antibodies, antihemophilic factors, interferon, vaccines, enzymes. Agriculture: production of Golden Rice First… Bacteria in 1973 Mice in 1974 Insulin-producing bacteria in 1982 Gene therapy in 1990 Genetically modified food has been sold since 1994 Trangenic animals • are useful for research of gene therapy and human pathologic genotypes and phenotypes • expression of mammals genes • model organism for testing vectors Transgenic mice, sheeps, pigs, cattle…. hens, sheep, goats, rabbits, pigs, cattle with resistance to diseases, to enhanced quality of meat and milk Methods important for medical sciences: Production of monoclonal antibodies VNTR variable number of tandem repeats Single nucleotide polymorphisms SNPs Allele specific oligonucleotide analysis (ASO) Microarrays - Identifying sets of disease genes Pharmacogenomics Nanomedicine - may be used for delivery of small sensors to target sites in body, detect and destroy cancer cells Monoclonal antibodies Microarrays Dr. Peter Snustad, Michael J.Simmons: Principles of Genetics, 5. edition, 2009 http://www.clinigene.eu/video-intro-gene-therapy.html Thank you for your attention Commercial cloning Genetic donor Somatic nucleus transfer 1 clon 32 000 USD (r.2004) Deposition of animal cells into bank 850 USD (r.2005) clones gizmo malý gizmo tahini nicky baba nanusch a tabouli malý nicky