Concepts and Methods in Developmental Biology

Transcription

1 Biology 4361 Developmental Biology Concepts and Methods in Developmental Biology June 16, 2009

2 Conceptual and Methodological Tools Concepts Genomic equivalence Differential gene expression Differentiation/de-differentiation Technical Methods Cloning RNA localization techniques RT-PCR microarrays macroarrays in situ hybridization Transgenics microinjection chimeras embryonic stem cells Gene knockouts, etc. Antisense RNA RNA interference

3 Differentiation Overview Differentiation cells become structurally and functionally different Selective expression of certain genes = production of specific proteins Specific proteins = specialized cell Differentiation: one-way (i.e. normally, no de-differentiation) Detecting differentiation (or stages): differential gene expression mrna DNA transcription protein whole cell techniques

4 Concept: Genomic Equivalence Every cell in an organism has the same set of genes. If so, shouldn t any nucleus be able to direct the development of any other cell, even the zygote? Test: somatic nuclear transplantation (cloning) - use a somatic (i.e. differentiated) nucleus to direct development Technical challenges: - enucleation of a recipient egg - acquisition of competent donor nuclei - ability to transfer nuclei into enucleated oocytes

5 Method - Amphibian Cloning (activates egg)

6 Effect of Age in Nuclear Transplants Transplant nuclei from different aged donors. Rana pipiens Loss of ability of nuclei to support development = loss of blastomere potency

7 Serial Transplantation Clones made with differentiated nuclei died shortly after the blastula stage. However, if blastomeres from cloned blastulae were used as nuclear donors. and the process was repeated several times. clones could survive. Xenopus laevis Differentiated nuclei still had pluripotent potential but de-differentiation was necessary

8 Method - Mammalian Cloning Udder cell - differentiated

9 Mammalian Cloning - 2 Inner cell mass (ICM) forms embryo proper Trophoblast forms connections with placenta Mammalian cloning success in multiple species, however - inefficient (Dolly = 1/434 transfers) Problems: - large fetus - old chromosomes - disease susceptibility - premature aging

10 Concept: Differential Gene Expression Changes seen in blastomeres as they age = changes in the expression of genes, not in the content of the genome. Differential gene expression is the key to changing phenotype of the cell.

11 Concept: Differential Gene Expression Three postulates of differential gene expression: 1. Every nucleus contains the complete genome; DNAs of all differentiated cells are identical. 2. Unexpressed genes in differentiated cells are not destroyed or mutated, but retain the potential for being expressed. - during development, cells constantly change function - serial nuclear transplantation showed re-programming of differentiated cells 3. Only a small percentage of the genome is expressed in each cell. - polytene chromosomes illustrate expression patterns

12 Polytene Chromosomes (Drosophila) Illustrate genomic equivalence and differential gene transcription: - banding patterns same in different cell types (i.e. same chromosomes) - however, patterns of transcriptional activity are different

13 Chromosome Puffs Active Transcription Puffs represent active gene transcription Claus Pelling, Max-Planck Institute of Biology, Tübingen. control + actinomycin (prevents RNA synthesis) - changing patterns over time = regulation of transcriptional activity Timed sequence of Drosophila chromosome puffs; ~ 20 h Michael Ashburner, University of Cambridge

14 Method - Transgenic Cells and Organisms Transgenics move genes in (or out) of organisms Why?

15 Method - Chimera Formation Mammalian blastocyst inner cell mass - embryo proper trophoblast - embryonic contribution to the placenta insert gene into embryo using - transfection - microinjection - etc. NOTE - vector contains antibiotic resistance gene

16 - and some may integrate into the germ line (gamete progenitors) Method - Chimera Formation ES cells (with transgene and antibiotic resistance gene) NOTE growth medium contains antibiotic!

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18 mouse (R) - transgenic for human growth hormone R. L. Brinster and R. E. Hammer

19 Method - Gene Knockouts Targeted gene insertion - similar to chimeric method, but.. - selectively replaces functional genes with non-functional genes target gene: bone morphogenic protein 7 (BMP7) mutation: insert a gene for neomycin resistance into the BMP7 gene regions of homology - neo r = neomycin resistance

20 The presence of the antibioticresistance gene allows selection of only successfully incorporated knockout genes

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22 Method - Antisense RNA mrna single stranded message = sense strand: 5 C-A-U-G3 complement (synthesized) = antisense strand: 3 G-U-A-C5 Insertion of antisense RNAs into the cell blocks gene expression: - translational blockage (synthetic antisense RNAs) - transcriptional blockage - postulated function of natural antisense RNAs

23 Methods Method - Antisense RNA Synthesis and insertion of antisense RNA - binds to native mrna - double stranded RNA subject to enzymatic destruction (Dicer) Morpholino antisense oligomers - morpholino molecules used in strand construction - morpholino oligomers do not degrade; effective over several cell generations morpholino Note - naturally-occurring antisense RNAs also exist: - paternal gene for IGF2r receptor contains antisense sequence - blocks synthesis of IGF2r

24 Method - RNA-Interference RNA interference (RNAi) - double-stranded RNAs inserted (short interfering RNA (sirna) - leads to the degradation of native mrna - RNAi is common in plants, animals, fungi - probably evolved as a defense against retroviruses = dsrnas

25 Method - RNA-Interference Dicer enzyme complex binds to dsrna - cleaves into short interfering RNA (sirna) Note - when dsrna is from endogenous source = microrna (mirna) RISC (RNA induced silencing complex) - argonaut component of Dicer complex catalyzes mrna degradation

26 RNAi mechanisms triggers: RNAi Knockdown RNAi-inhibited transcription: knockdown experiments - blocks transcription of specific genes - lack of gene = lack of protein = lack of function - advantage: effect can be seen at certain time in certain tissue - like knockouts, but not as complete - sirnas specific for particular sequence - mirnas less specificity mirna stem-loop sirna dsrna, probably from RNA viruses aka short hairpinrna (shrna) microrna (mirna) - genetically coded sequences; - non-coding; - control some translation during portions of development - extensive post-transcriptional modification