Molecular Approaches for the Analysis of Gene Structure and Function

1. Cardiac adrenoreceptors: model genes

2. Cultured primary cells: heart cells

3. Heterologous cultured cells: model system to study gene function

4. Detection of expressed proteins: anti-sera to native protein or epitope tagging

5. Detection of Protein-protein interactions: Immunoprecipitation, BRET and yeast 2-hybrid

6. The use of Green Fluorescent protein (GFP)

7. RNAi technology: knock down of endogeneously expressed genes

8. Animal models: transgenics and gene knockouts

1.Cardiac adrenoreceptors: model genes

Copyright ©1999 by the National Academy of Sciences

Limbird, Lee E. and Vaughan, Douglas E. (1999) Proc. Natl. Acad. Sci. USA 96, 7125-7127

Receptor signalling in the healthy and in the failling heart

2. Culture of animal cells Cells are dispersed from tissues and are cultured in vitro. Most primary cells do not grow but can be kept in nutrient media for days to weeks.

Cells in culture. (A) Phase-contrast micrograph of fibroblasts in culture. (B) Micrograph of myoblasts in culture shows cells fusing to form multinucleate muscle cells. (C) Oligodendrocyte precursor cells in culture. (A, courtesy of Daniel Zicha; B, courtesy of Rosalind Zalin; C, from D.G. Tang et al., J. Cell Biol. 148:971  984, 2000)

2.Cultured primary cells: heart cells

Cultured cells can be used to study the different effects of “Growth stimuli” such as adrenergic agonists. Heart cells express all 3 Beta adrenergic receptor. To study individual receptors, heterologous cells are often used.

J Biol Chem. 2000 Nov 3;275(44):34528-33

Iso (β-AR agonist) induces cardiomyocyte apoptosis. Cardiac myocytes were incubated for 48 h with the indicated concentrations of Iso (0.1-50 µM) or 1 µM ionomycin. The percentage of TUNEL-positive cardiomyocytes is presented as the average ± S.E. from three independent experiments. *, p < 0.05 versus control.

Cannot study individualβARs in cultured heartcells. Use heterologouscells.

CELL LINE* CELL TYPE AND ORIGIN

3T3 fibroblast (mouse)

BHK21 fibroblast (Syrian hamster)

MDCK epithelial cell (dog)

HeLa epithelial cell (human)

PtK1 epithelial cell (rat kangaroo)

L6 myoblast (rat)

PC12 chromaffin cell (rat)

SP2 plasma cell (mouse)

COS kidney (monkey)

293 kidney (human); transformed with adenovirus

CHO ovary (chinese hamster)

DT40 lymphoma cell for efficient targeted recombination (chick)

R1 embryonic stem cells (mouse)

E14.1 embryonic stem cells (mouse)

H1, H9 embryonic stem cells (human)

S2 macrophage-like cells (Drosophila)

Some Commonly Used Cell Lines**

**Many of these cell lines were derived from tumors. All of them are capable of indefinite replication in culture and express at least some of the special characteristics of their cell of origin. BHK21 cells, HeLa cells, and SP2 cells are capable of efficient growth in suspension; most of the other cell lines require a solid culture substratum in order to multiply.

3.Hetero-logousCells

Introduction of DNA into animal cells A eukaryotic gene of interest is cloned in a plasmid containing a drug resistance marker that can be selected for in cultured animal cells. The plasmid DNA is introduced into cultured cells as a calcium phosphate coprecipitate, which is taken up and expressed by a fraction of the cells for a few days (transient expression). Stably transformed cells, in which the plasmid DNA becomes integrated into chromosomal DNA, can be selected for by their ability to grow in drugcontaining medium.

Individual βAR canbe introduced into cells and studied.

Fusion proteins are often designed as immunogens for raising antibodies. In this example, the plasmid vector has an origin of replication (ori) and an ampicillin resistance gene (ampr) for growth in E. coli. The multiple cloning site (MCS) is located immediately adjacent to a lacZ gene which can encode -galactosidase with transcription occurring from the lacZ promoter (PLAC) in the direction shown by the arrow. A cDNA sequence from a gene of interest (gene X) is cloned in a suitable orientation into the MCS. Expression from the lacZ promoter will result in a -galactosidase-X fusion protein. This can be used as an immunogen to raise antibodies to protein X. A popular alternative is to use GST fusion proteins, where glutathione S transferase is coupled to the protein of interest. The fusion protein can be purified easily by affinity chromatography using glutathione agarose columns.

Purify proteinto use as antigen (or for functionalassays)

4. Detection of expressed Proteins: anti-sera to native protein

Preparation of Monoclonal Antibodies. Hybridoma cells are formed by fusion of antibody-producing cells and myeloma cells. The hybrid cells are allowed to proliferate by growing them in selective medium. They are then screened to determine which ones produce antibody of the desired specificity. [After C. Milstein. Monoclonal antibodies. Copyright © 1980 by Scientific American, Inc. All rights reserved.]

Polyclonal Ab are present in the sera of animals (often rabbits) that are injected with the antigen (immunogen)

Western blotting Proteins are separated according to size by SDS-polyacrylamide gel electrophoresis and transferred from the gel to a filter. The filter is incubated with an antibody directed against a protein of interest. The antibody bound to the filter can then be detected by reaction with various reagents, such as a radioactive probe that binds to the antibody.

Detection of primary Ab with conjugated 20 Ab

Diabetes 50:455-461, 2001

Quantitation of the amount of ß1-, ß2-, and ß3-AR in plasma membrane preparations from control ( ), 14-week—STZ-induced diabetic ( ), and 12-week—STZ-induced/2-week—insulin-treated diabetic ( ) rat hearts.

Western blot analysis can be used to determine the expression of specific proteins

Epitope tagging allows the localization or purification of proteins. Using standard genetic engineering techniques, a short epitope tag can be added to a protein of interest. The resulting protein contains the protein being analyzed plus a short peptide that can be recognized by commercially available antibodies. The labeled antibody can be used to follow the cellular localization of the protein or to purify it by immunoprecipitation or affinity chromatography.

4. (continued) Detection of expressed Proteins: epitope tagging

Epitope tagged proteins canbe detected using Abs directed against epitope. No need for Ab toendogenous protein.

Commonly used epitope tags

Sequence of tag Origin Location mAb

DYKDDDDK synthetic FLAG N, C terminal anti-FLAG M1

EQKLISEEDL human c-Myc N, C terminal 9E10

MASMTGGQQMG T7 gene 10 N terminal T7.Tag Ab

QPELAPEDPED HSV protein D C terminal HSV.Tag Ab

RPKPQQFFGLM substance P C terminal NC1/34

YPYDVPDYA influenza HA1 N, C terminal 12CA5

Figure 1 | Role of homo- and heterodimerization in the transport of G-protein-coupled receptors.   When expressed alone, the GABABR1 (GBR1) receptor is retained as an immature protein in the endoplasmic reticulum (ER) of cells and never reaches the cell surface. By contrast, the GBR2 isoform is transported normally to the plasma membrane but is unable to bind GABA and thus to signal. When coexpressed, the two receptors are properly processed and transported to the cell surface as a stable dimer, where they act as a functional metabotropic GABAB receptor.

Nat Rev Neurosci. 2001 Apr;2(4):274-86

GPCR dimerization

5.Detection of Protein-protein interactions: Immunoprecipitation, BRET and 2-hybrid

Figure 19.4.1 Flow chart for the coprecipitation of two proteins that have been differentially tagged and introduced into the host organism. Ig h and Ig l, immunoglobulin heavy and light chains; NT, no tag.

Current Protocols in Protein SciencePublished by John Wiley & Sons, Inc

Fig. 1. Principle of bioluminescence resonance energy transfer assay. Upper panels: Schematic representation of seven transmembrane opioid receptors fused to Renilla luciferase (Rluc) and yellow fluorescent protein (YFP). When they are far apart the light from the luminescent donor Rluc cannot excite the fluorescent acceptor YFP (A). If two differentially tagged receptors interact and are brought close together the acceptor is excited and emits light at 530 nm (B). Lower panels: Typical spectra obtained in the absence (A) and in the presence (B) of receptor–receptor interactions with a peak at 470 nm (A) and

peaks at 470 and 530 nm (B).

The yeast two-hybrid system for detecting protein-protein interactions. The target protein is fused to a DNA-binding domain that localizes it to the regulatory region of a reporter gene as "bait." When this target protein binds to another specially designed protein in the cell nucleus ("prey"), their interaction brings together two halves of a transcriptional activator, which then switches on the expression of the reporter gene. The reporter gene is often one that will permit growth on a selective medium. Bait and prey fusion proteins are generated by standard recombinant DNA techniques. In most cases, a single bait protein is used to fish for interacting partners among a large collection of prey proteins produced by ligating DNA encoding the activation domain of a transcriptional activator to a large mixture of DNA fragments from a cDNA library.

Schematic diagram representing key steps in GPCR signaling and homologous desensitization. Note that the receptor gets internalized in an agonistDependant manner.

6. The use of Green Fluorescent protein (GFP)

GFP allows for thedetection of proteinmovement within a cell

Translocation of ß-arrestin 2-GFP to the ß2-adrenergic receptor (ß2AR). HEK

293 cells stably overexpressing the ß2AR were transiently transfected with ß-

arrestin 2-GFP. The distribution of ß-arrestin 2-GFP fluorescence was visualized by confocal microscopy before (−Iso) and after a 5 min treatment with isoproterenol (+Iso; 10−8, 10−6 M) at 37 °C. Before agonist-stimulation, ß-arrestin 2-GFP is uniformly distributed throughout the cytosol. Upon agonist addition, ß-arrestin 2-GFP translocates from the cytosol to the plasma membrane where it is found colocalizing with the receptor in punctuated areas of the plasma membrane.

Cytoskeleton dynamics in living cells, as illustrated by changes in the microtubule and actin filament network during cell spreading and the rearrangement of stress fibres after cyclic stretching.

Nature Materials 2, 715–725 (2003)

The Mechanism of RNA Interference (RNAi)

Ambion

7. RNAi technology: knock down of endogeneously expressed genes

RNAi

Introducing RNAi into cells.

RNAi allows the study of cellslacking a specific protein (gene)

RNAi-mediated knockdown of Cav-1. A, representative immunoblot (IB) of five clonally isolated cell lines screened for the presence of Cav-1 (equal amount of protein was loaded in duplicates). B, representative immunoblot for RNAi clone showing knockdown of protein Cav-1 with respect to wt C6 glioma cell lysates. Samples were loaded in duplicate and also probed for total G q and RSK1 to assess nonspecific knockdown of unrelated proteins.

Example of RNAi knock down.

Knockdown of Cav-1 expression impairs signaling of 5-HT2A receptors. For these experiments wt C6 glioma cells and RNAi-mediated knocked down Cav-1 cells were plated onto 96-well plates. Panel shows the sigmoid dose response to serotonin (5-HT) in normalized relative fluorescence units (RFU).

Gene replacement, gene knockout, and gene addition. A normal gene can be altered in several ways in a genetically engineered organism. (A) The normal gene (green) can be completely replaced by a mutant copy of the gene (red), a process called gene replacement. This provides information on the activity of the mutant gene without interference from the normal gene, and thus the effects of small and subtle mutations can be determined. (B) The normal gene can be inactivated completely, for example, by making a large deletion in it; the gene is said to have suffered a knockout. (C) A mutant gene can simply be added to the genome. In some organisms this is the easiest type of genetic engineering to perform. This approach can provide useful information when the introduced mutant gene overrides the function of the normal gene.

8. Animal models: transgenics and gene knock-outs

Figure 8-36. General procedure for producing transgenic mice. [See R. L. Brinster et al., 1981, Cell 27:223.] Molecular Cell Biology

Summary of the procedures used for making gene replacements in mice. In the first step (A), an altered version of the gene is introduced into cultured ES (embryonic stem) cells. Only a few rare ES cells will have their corresponding normal genes replaced by the altered gene through a homologous recombination event. Although the procedure is often laborious, these rare cells can be identified and cultured to produce many descendants, each of which carries an altered gene in place of one of its two normal corresponding genes. In the next step of the procedure (B), these altered ES cells are injected into a very early mouse embryo; the cells are incorporated into the growing embryo, and a mouse produced by such an embryo will contain some somatic cells (indicated by orange) that carry the altered gene. Some of these mice will also contain germ-line cells that contain the altered gene. When bred with a normal mouse, some of the progeny of these mice will contain the altered gene in all of their cells. If two such mice are in turn bred (not shown), some of the progeny will contain two altered genes (one on each chromosome) in all of their cells. If the original gene alteration completely inactivates the function of the gene, these mice are known as knockout mice. When such mice are missing genes that function during development, they often die with specific defects long before they reach adulthood. These defects are carefully analyzed to help decipher the normal function of the missing gene

Stimulation of β1-AR but not β2-AR increases DNA fragmentation, assayed by DNA laddering (A) or nucleosomal ELISA (B). *, P < 0.01 versus untreated (Sta or ISO) or uninfected myocytes (DKO and Fresh) and those infected by adeno- β2-AR (n = 4-6 for each group).

PNAS |2001 | vol. 98 | no. 4 | 1607-1612

Culturing cardiomyocytes from mice lacking both βARs allows one to re-introduce individual βARs and ascertain their specificfunction.

Using DNA microarrays to monitor the expression of thousands of genes simultaneously. To prepare the microarray, DNA fragments   each corresponding to a gene   are spotted onto a slide by a robot. Prepared arrays are also available commercially. In this example, mRNA is collected from two different cell samples for a direct comparison of their relative levels of gene expression. These samples are converted to cDNA and labeled, one with a red fluorochrome, the other with a green fluorochrome. The labeled samples are mixed and then allowed to hybridize to the microarray. After incubation, the array is washed and the fluorescence scanned. In the portion of a microarray shown, which represents the expression of 110 yeast genes, red spots indicate that the gene in sample 1 is expressed at a higher level than the corresponding gene in sample 2; green spots indicate that expression of the gene is higher in sample 2 than in sample 1. Yellow spots reveal genes that are expressed at equal levels in both cell samples. Dark spots indicate little or no expression in either sample of the gene whose fragment is located at that position in the array. For details see Figure 1-45. (Microarray courtesy of J.L. DeRisi et al., Science 278:680  686, 1997. © AAAS.)