Genetic Manipulation

Data

Official data in SubjectManager for the following academic year: 2024-2025

Topic

The aim of the course is to present the theoretical and practical background of nucleic acid manipulation and vector-design. The course discusses in detail the methods and aims of nucleic acid manipulation and also the establishment of recombinant viral vectors. Participants will become familiar with the practical utilization of vectors, the genetic manipulation of cells and laboratory animals, and how e.g. a gene-therapeutic intervention is designed in the case of a monogenic recessive hematological disorder.

Lectures

Practices

• 1. Rules of primer design, oligonucleotide adaptor design, cloning primer design.
• 2. Performing cloning PCR. EtBr gel-electrophoresis to evaluate PCR cloning efficiency.
• 3. Getting familiar with plasmid restriction endonuclease maps. Planning and performing restriction endonuclease digestion of plasmid DNA.
• 4. Gel-electrophoresis and evaluation of restriction mapping. Cutting gel pieces containing favored DNA.
• 5.

  Isolation of favored DNA from gel pieces. Ligation of insert and plasmid DNA fragments. Heat shock transformation of chemically competent E. coli. Plating transformed bacteria over antibiotic plates.

  Verification / analysis of E. coli hosts containing plasmid construct of interest using colony PCR.

• 6. Gel electrophoresis and evaluation. Initiation of bacterial cultures for the propagation of plasmid constructs of interest.
• 7. Harvesting bacterial cultures initiated for plasmid propagation. Isolation of plasmids using commercial mini-prep kits. Measurement of plasmid purity and quantity.
• 8. Verification of plasmid identity using restriction endonuclease digestion and gel electrophoresis.
• 9. Transient transfection of adherent cell lines with marker gene and transgene of interest.
• 10. Evaluation of transfection efficiency with fluorescent flow cytometry.
• 11. Isolation of DNA and RNA from transgenic cells. Measurement of DNA and RNA isolate purity and quantity.
• 12. Initiation of cDNA synthesis using recombinant reverse transcriptase
• 13. Real-time quantitative PCR assembly, real-time follow-up and evaluation. (Q-PCR primers specific for transgene and marker gene)
• 14. Histological staining of control and transgenic cell cytospin slides using transgene-specific fluorescent antibodies and marker gene.
• 15. Evaluation of co-localization with marker gene expression.
• 16. Transient transfection of adherent cell lines with RNAi probes.
• 17. Evaluation of transfection efficiency with fluorescent microscopy.
• 18. Histological staining of control and RNAi-treated cell cytospin slides using gene-specific fluorescent antibodies I.
• 19. Histological staining of control and RNAi-treated cell cytospin slides using gene-specific fluorescent antibodies II.
• 20. Live visualization of transgenic mice.
• 21. Histological evaluation of 3D transgene expression pattern using Z-stack technology
• 22. Cutting gel pieces containing favored DNA
• 23. Isolation of plasmids using commercial mini-prep kits.
• 24. Heat shock transformation of chemically competent E. coli.
• 25. Short presentations of students
• 26. Short presentations of students
• 27. Summary
• 28. Test

Seminars

• 1. Revision of basic molecular biology: DNA and gene structure, flow of genetic information
• 2. Analysis of genome, transcriptome, genetic polymorphisms
• 3. Basic DNA manipulation toolkit I: restriction endonuclease digestion, ligation
• 4. Basic DNA manipulation toolkit II: DNA modification, labeling, hybridization
• 5. What is gene cloning and how is it performed?
• 6. Verification and characterization of cloned genes and their products (DNA, RNA, protein level)
• 7. Genomic and cDNA libraries (random, arrayed and ordered libraries)
• 8. Conventional gene knock-out technologies, applications and limitations
• 9. RNAi or mRNA-specific knock-down, applications and limitations
• 10. Temporal and spatial orchestration of gene expression / tissue and developmental stage specific promoters
• 11. Non-viral vectors and gene delivery techniques
• 12. Viral vectors, in vitro and in vivo applications
• 13. Lentiviral transgenesis
• 14. Artificial chromosomes

Reading material

Obligatory literature

Literature developed by the Department

Notes

Recommended literature

1. S. B. Primrose & R. M. Twyman: Principles of Gene Manipulation and Genomics, 7th edition, Blackwell Publishing. 2006. 626 pages. ISBN 1 405 13544 1

2. Pongracz J, Keen M (eds.): Medical Biotechnology, Elsevier, 2009

3. Tom Strachan: Human Molecular Genetics, Garland Publishing, 2004

Conditions for acceptance of the semester

Labnotes submitted by end of semester

Mid-term exams

Written test

Making up for missed classes

None

Exam topics/questions

www.medbiotech.com

Examiners

Instructor / tutor of practices and seminars

• Kvell Krisztián