Chapter 15: Genetically Modified Organisms: Use in Basic and Applied Research

1. Chapter 15: Genetically Modified Organisms: Use in Basic and Applied Research

2. Dolly is living proof that an adult cell can revert to embryonic stage and produce a full new being. This was not supposed to happen. Charles Krauthammer, Time (1997) 149:60

3. 15.1 Introduction

4. Genetically modified organisms are no longer the realm of science fiction…

5. Transgenic organism • Carries transferred genetic material (the transgene) that has been inserted into its genome at a random site. Knockout organism • Created by gene targeting—the replacement or mutation of a particular gene.

6. Cloned organism • A genetically-identical organism produced by nuclear transfer from adult somatic (body) cells to an unfertilized egg.

7. 15.2 Transgenic mice

8. 1980:the first transgenic mouse was produced by microinjection of foreign DNA into fertilized eggs. • 1982: “Super” mice expressing rat growth hormone gene coding sequence.

9. OncoMouse patent • Is a transgenic mouse an invention? • US patent for a mouse whose germ cells and somatic cells contain an activated oncogene sequence. • The patent remains controversial worldwide.

10. How to make a transgenic mice Three main stages in the process: • Microinjection of DNA into the pronucleus of a fertilized mouse egg. • Implantation of the microinjected embryo into a foster mother. • Analysis of mouse pups and subsequent generations for the stable integration and expression of the transgene.

11. Pronuclear microinjection • Transgene: What are the minimal requirements for expression of a cDNA? • Critical window of time before pronuclei fuse to form a diploid zygotic nucleus. • Usually inject the sperm pronucleus since it is larger and closer to the egg surface.

12. Implantation into foster mother • Manipulated embryos are transferred into a recipient “pseudopregnant” mouse. • Pregnancy is visible about 2 weeks after embryo transfer. • Litter is delivered about 1 week later.

13. Analysis of mouse pups Two important questions: • Is there stable integration of the transgene into the mouse chromosome. • If the transgene is present, is it expressed appropriately?

14. Analysis of stable integration • Success rate is ~2.5 to 6% in mice. • Tail biopsies for DNA analysis by Southern blot or PCR. • Integration is random and occurs by nonhomologous recombination. • More than one copy may be integrated.

15. Analysis of transgene expression At the level of transcription • Northern blots • RT-PCR • In situ hybridization, etc. At the level of translation • Western blots • Immunohistochemistry • GFP expression, etc.

16. Transposon tagging • Transposable elements have provided a powerful tool for insertional mutagenesis studies. • A method to link phenotype with genomic sequence.

17. Example: A transposon carrying antibiotic resistance is introduced into pathogenic bacteria. • Screen for nonfunctional mutants, which indicates that insertion of the transposon disrupted a gene important for pathogenicity.

18. Example: Gene knockout in mice by insertional mutagenesis using a “Sleeping Beauty” transposon. • The mouse strain already contains the Sleeping Beauty transposase. • Transposition activity is marked by activation of GFP at the new location.

19. Inducible transgenic mice • What can be done if the transgenic is embryonic lethal? • e.g. Inducible “Tet-off” expression system

20. 15.3 Gene-targeted mouse models

21. The ability to create a mouse of any desired genotype. • A US-based consortium is systematically knocking out mouse genes one by one in embryonic stem cells. • A European-based consortium is engineering knockout cells containing genes that can be switched on or off at any stage of development in the mutant mouse.

22. Knockout mice Five main stages: • Construction of the targeting vector. • Gene targeting in embryo-derived stem (ES) cells. • Selection of gene-targeted ES cells.

23. Introduction of ES cells into mouse embryos and implantation into a foster mother. • Analysis of chimeric mice and inbreeding to obtain a pure breeding strain of “knockout mice.”

24. The phenotype of the knockout mouse displays the impact of the targeted gene on development and physiology. • Example: Argonaute2 knockout mice show severe developmental delay.

25. Knockin mice • Often used for in vivo site-directed mutagenesis. • Mutant knockin allele replaces the coding region of the endogenous allele.

26. Knockdown mice • Analysis of cis-regulatory regions. • Knockdown targeting sequence disrupts endogenous upstream regulatory elements, while keeping the coding region intact.

27. Conditional knockout and knockin mice • Gene knockouts often result in embryonic lethality. • To study a gene’s role later in development, genetic switches such as the Cre/lox system are used.

28. Cre/lox system for site-specific recombination • Cre recognizes a 34 bp site on the bacteriophage P1 genome called lox. • Catalyzes reciprocal recombination between pairs of lox sites.

29. Inducible gene expression in mice using the Cre/lox system • Activation of transgene expression by site-specific recombination.

30. Conditional knockout by Cre-mediated recombination • Modify the target gene in ES cells so that it is flanked by lox sites. • Mice containing the modified gene are crossed with mice expressing Cre in the desired target tissue. • Cre-mediated excision results in tissue-specific gene knockout.

31. 15.4 Other applications of transgenic animal technology

32. Transgenic animals have been explored as tools for applied purposes, ranging from artwork to pharmaceuticals.

33. Transgenic artwork: the GFP bunny • Alba the GFP bunny was commissioned by artist Eduardo Kac.

34. Transgenic primates • Mice do not always provide an accurate model of human physiology and disease pathology. • Interest in extending transgenic and gene-targeting studies to nonhuman primates. • 2001: ANDi, the first transgenic rhesus monkey carrying the GFP transgene, did not glow green.

35. Transgenic livestock • Attempts to use pronuclear microinjection in large animals have had only limited success. • Development of linker-based sperm-mediated gene transfer (LB-SMGT) has greatly improved efficiency.

36. Gene pharming • Turning animals into pharmaceutical bioreactors for protein-based human therapeutics. • e.g. production of therapeutic proteins in milk or egg white.

37. 15.5 Cloning by nuclear transfer

38. The first animal cloning experiments were conducted in the 1950s in the leopard frog, Rana pipiens. • Briggs and King were interested in directly testing the question of genetic equivalence of somatic cell nuclei.

39. Genetic equivalence of somatic cell nuclei: frog cloning experiments • Long-standing question in developmental biology: • Does cell differentiation depend on changes in gene expression or changes in the content of the genome?

40. Nuclear transplantation experiments in Rana pipiens and Xenopus laevis showed that some normal adult frogs could develop from the nuclei of differentiated cells. • In general, cell differentiation depends on changes in the expression not content of the genome.

41. Cloning of mammals by nuclear transfer • A major challenge in performing somatic cell nuclear transfer in mammals is the small size of the mammalian egg. • Transfers of nuclei from very early embryos to enucleated sheep eggs were not successfully performed until 1986. • Cloning attempts of nonhuman primates have proved even more difficult.

42. “Breakthrough of the year:” the cloning of Dolly • Dolly was the first mammal cloned from an adult cell. • Less the 1% of all nuclear transfers from adult differentiated cells result in normal-appearing offspring.

43. The cloning of Dolly confirmed two key principles of genetic equivalence: • Differentiated cells on their own are unable to develop into complete animals but the nuclei of most differentiated cells retain all the necessary genetic information. • Transfer of a nucleus from a differentiated cells to the environment of the enucleated egg reprograms the nucleus and allows full development.

44. Method for cloning by nuclear transfer Four main steps: • Preparation of donor cells. • Enucleation of unfertilized eggs. • Nuclear transfer by cell fusion.

45. Implantation of the embryo into a surrogate mother and analysis of clones. • DNA typing techniques can be used to confirm that the cloned offspring is genetically identical to the original donor cell nucleus.

46. Source of mtDNA in clones • When the cell fusion method is used, the reconstructed embryo will contain egg cytoplasm and the donor nucleus with its accompanying cytoplasm. • The clone will be heteroplasmic for mtDNA.

47. Why is cloning by nuclear transfer inefficient? • To create Dolly, it took 277 trials. • When 10,000 genes were screened in cloned mice, 4% were shown to be functioning incorrectly. • Cloned animals suffer from many developmental abnormalities.

48. Inefficient reprogramming of the genome. • Effects of cellular aging. • Improper segregation of chromosomes during embryonic cell divisions.

49. Example: • Rhesus monkey embryos generated by nuclear transfer. • Missing important components of the mitotic spindle.

50. Reprogramming the genome • Totipotent cells are capable of forming any cell type. • Pluripotent cells are capable of differentiating into several different cell types. • Differentiated cells are specialized towards a specific function by differential gene expression.