- •2) Objects and methods of animal biotechnology
- •3) Totipotent, multipotent, pluripotent animal cells
- •5)The principles of genetic cloning
- •6.Allophenic animals. Genetic chimers
- •8) Methods for introducing foreign dna into animal cells
- •9)Cryopreservation of reproductive and germ cells of animals and humans
- •11)The principles and methods of plant cells cultivation in vitro
- •14)Differentiation and dedifferentiation in plant cell culture. The obtaining callus mass and cultivation of callus tissue .
- •15)The influence of phytohormons on morphogenesis and regeneration in plant cells culture
- •16.The main path of morphogenesis processes in plant cells culture
- •20) The factors influenced on microclonal propagation in plant cell culture.
- •21) What is Biotechnology? Various definitions of “Biotechnology”. History of Biotechnology
- •22.Microbial Biotechnology: fundamentals of applied microbiology
- •26) Somaclonal and gametoclonal variation in plant cells culture.
- •27) Artificial seeds". Embryo culture in vitro
- •28. Culture of apical meristem cells
- •29)Cell reconstruction. Theoretical means of cell reconstruction
- •32) Main objects of animal biotechnology:
- •33) Morphological and functional features of gametes - eggs and sperm
- •39) Basic approaches and principles of gene therapy. Gene therapy ex vivo, in vivo, in situ.
- •35)The history of investigations of the genetic transformation of animal cells
- •53)Genetic engineering. Methods of genetic transformation
- •56) The vector systems used in the genetic engineering
- •62) Methods of producing chimeras
- •57) Methods of genetic engineering: agrobacterial genetic transformation
- •63) Collection and cultivation of oocytes in vivo and in vitro
- •68) Draw a diagram of the structure of plasmid pBr322
- •69) Draw a diagram of an experiment in genetic engineering (design recDna) and give a description of the main stages
- •74) Modes of freezing and thawing of gametes and embryos
- •75) Methods of artificial fertilization: gamete insemination fallopian tube (gift), zygosity insemination fallopian tubes (zift).
- •80) Methods of animal cloning, reproductive and therapeutic cloning
- •81) Microorganisms in water and wastewater treatment
- •86) Use of e.Coli for the biotechnological production
- •87) Microbes in milk and dairy products
56) The vector systems used in the genetic engineering
Host-Vector Systems
Cloning of foreign DNA (in plasmid or phage vectors)
Subsequent manipulations often require vectors that can deliver a cloned segment into eukaryotic cells (e.g. - to understand the mechanism of gene expression in a eukaryotic cell). A number of vectors have been devised to satisfy this need. They often contain pieces of eukaryotic viruses to facilitate entry into the cell and expression or integration once in the cell itself.
Note that each small extrachromosomal genome (phage, plasmid, or eukaryotic virus) is found in nature within a particular species and replicates only within cells of its natural host or within cells of closely related species. The fundamental tool is, therefore, a two component system: a host-vector system.
Expression and Shuttle Vectors
Expression vectors allow you to express certain genes directly from their recombinant DNAs. A typical expression vector will have a promoter upstream of the DNA containing the sequence to be expressed. Usually the "gene" is a cDNA because if the gene contained introns the introns would not be removed in bacteria. In addition, the promoter that is used can be an inducible one, so that synthesis of the gene product can be regulated. For example, if you wanted to express human growth hormone, you cut the DNA with an appropriate restriction enzyme to isolate the growth hormone. Then open the vector with the same restriction enzyme, allowing you to place the growth hormone cDNA downstream of a lac promoter. In the presence of lactose, bacteria containing this construct will produce human growth hormone.
This approach has made available many reagents that were not available before such as tissue plasminogen activator for the treatment of heart attacks, erythropoietin for treatment of anemia, interferons for treatment of cancer and hepatitis infections, human insulin for diabetes, and many others to come.
Shuttle vectors allow DNA to be transferred between two different species. The shuttle vector has two origins of replication, allowing replication to occur in either system/host; it "shuttles" between two different species. Typically, one host is bacterial (e.g. E. Coli) and the other host is a eukaryotic organism (e.g. human). The bacterial host is used for all of the cloning steps and the eukaryotic host can be used to study the expression from that cloned gene or can be used to synthesize a product from the gene.
Reverse Genetics
Shuttle vectors can be used to perform what has been called reverse genetics. It is possible to replace or alter the sequence of regulatory elements that control expression of a given gene -- then put gene back into their normal host cells to see how gene behavior has changed. This provides information on how the regulatory element might function and which are the important sequences within the regulatory element itself. Our understanding of promoters was brought about through this kind of study.