The Genetic Core of Development - Biology | Outline | BIOL 411, Study notes of Developmental biology

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Chapter 4: The genetic core of development

I. What do genes have to do with how an organism progresses

from fertilized egg to adult?

A. Genomic equivalence

B. differential gene expression

Genomic equivalence = that every cell has the same nuclear genes **so how do cells perform specific functions and acquire diff. fates? Use cloning techniques to determine if/when nuclear potency is restricted: ---transferred nucleus into egg from a donor cell (eg. a differentiated muscle cell), can compare results from using donor cells at diff. dev. stages

a. Northern blotting

- can determine when (dev. stage) and where (tissue) different

mRNAs are expressed Protocol:

1. Extract RNA
2. Separate by size w/ gel electorphoresis
3. Transfer RNAs to nitrocellulose membrane and incubate with radioactive ssDNA from gene or interest
4. Results: if mRNA for gene is in sample the labeled DNA will bind and be detected by autoradiography

b. Reverse transcriptase polymerase chain reaction

(RT-PCR)

First, quick review of PCR amplification:

In developmental biology, DNA microarrays have already been used to:

1. compare gene expression for the entire Drosophila larva during metamorphosis: White KP, Rifkin SA, Hurban P, Hogness DS. 1999. Microarray analysis of Drosophila development during metamorphosis. Science 286: 2179-84.
2. look at which genes are upregulated or downregulated when embryonic stem cells are caused to differentiate in culture: Kelly DL, and Rizzino A. 2000. DNA microarray analyses of genes regulated during the differentiation of embryonic stem cells. Mol Reprod Dev. 56: 113-123.
3. compare the gene expression in activated vs. unactivated B and T cells: Ollila J, and Vihinen M. 1998. Stimulation of B and T cells activates expression of transcription and differentiation factors. Biochem Biophys Res Commun. 249: 475-

c. Microarrays

-to determine changes in transcription of many

genes

Procedure for microarray analysis:

1. Isolate mRNA from organism at one stage (eg. gastrula;

or from one cell type), use reverse transcriptase to

generate cDNA and label with green fluorescence.

2. Isolate mRNA from organism at another stage (eg.

neurula; or second cell type), use RT to generate cDNA

and label with red fluorescence.

3. Add (hybridize) both cDNA probes to chip (has all cDNA

of genome) and wash.

d. in-situ hybridization

Hans S et al., BMC Dev Biol, 2007

1. generate antisense mRNA from a target cloned gene where the gene is reversed, so the product transcribed is complementary to the gene’s normal product
2. antisense mRNA is labeled
3. cells labeled contain the mRNAs of the target gene

d. in-situ hybridization

a. Transgenic cells/organisms

1. Introducing genes into cells to study their function

---use microinjection to introduce cloned gene into cell

---can also use transfection to incorporate DNA into cells,

the cells take up the DNA

---or use electroporation to force DNA into cells w/

electrical current

Roszko, I et al., Dev Biol , 2006

a. Transgenic cells/organisms

1. Introducing genes into cells to study their function

---transposable elements = mobile DNA that jump around in genome, interupt genes and can use to study normal gene function, and can use for inserting genes in ectopic regions of genome Examples:

1. P-elements in Drosophila
2. T-DNA in Arabidopsis

a. Transgenic cells/organisms

3. Gene “knockout”

---disrupt at transcriptional level

Knockout phenotype examples:

b. Functions of the gene message

2. RNA interference

---use dsRNA to degrade the targeted message, does NOT inhibit translation

1. can inject dsRNA into animal.
2. These dsRNAs activate the enzyme, Dicer, that cuts the RNA into small pieces
3. The siRNAs bind to the RISC complex that unwinds the strands so that the siRNAs can bind the complementary endogenous RNAs, which are degraded Motegi and Sugimoto, Nat Cell Biol , 2006