Term 2 Lecture 8: Transgenics and reporter genes

Question 1

creating transgenic organisms - genetic transformation

Answer 1

Transformation: the insertion of recombinant DNA into host cells (aka transfection if host is an animal)
- the genetically altered host cell/ animal is transgenic
- 2 ways to create transgenics
- 2 types of transgenic organism:
stable transformants - genetic modifications are hereditable
transiently transformed - only specific organism affected ( sometimes for a limited time period)

Question 2

Method 1: Chemical

Answer 2

• treat cells with chemicals to make membranes more permeable to DNA using:
  a) calcium chloride ( for E.coli and other bacteria)
  b) lithium chloride ( for yeast and other single cell eukaryotes)
• this treatment makes the cells ‘competent’ they must then be incubated on ice for 20 mins and heat shocked at 42 degrees c
  c) for mammalian cells lipid solutions are used to form lipid micelles containing DNA. they fuse to the mammalian cell membranes releasing DNA into the cells e.g. for a mouse the DNA micelles would be mixed with embryonic or stem cells to be injected into a developing embryo and then into a female mouse to grow.

Question 3

Method 2: microinjection

Answer 3

• effective for C. elegans and drosophila embryos, can also be done with mouse eggs
• DNA introduced this way can be plasmid or linear

Question 4

Method 3: Leaf infiltration

Answer 4

• solution to transform the leaf is put into a syringe without a needle and the blunt tip is pressed against the leaf while it is emptied
• this pushes the DNA into the intracellular space
• DNA introduced by this method is carried by a bacterial vector

Question 5

Method 4: floral dipping

Answer 5

• effective in Arabidopsis and some other crops
• seedlings grown in netted pots that hold them in place
• when they start to produce flower buds they are turned upside down and dipped into a solution containing bacterial vectors carrying the DNA to be transferred
• the vectors are taken up into the flower buds and the DNA goes into the bud tissue and the developing pollen
• this stably transforms the plants ( creates hereditable modifications)

Question 6

why would you want to produce transient gene expression?

Answer 6

• results are possible in 1-4 days - very quick in comparison to stable transformation which involves generating seeds or breeding animals
  -mRNA can be used resulting in expression within minutes
• quick, small scale production of recombinant proteins
  -often a rapid way to check if gene expression vector is correct ^ protein is produced so you can be sure that there are no errors in the open reading frame before creating stable transformants
• in vivo protein-protein/ protein interaction studies can be carried out
• useful if you’re not interested as to where the gene is expressed but how the proteins interact

Question 7

Transient vs stable transformation

Answer 7

Transient:
- transfer DNA not integrated into genome but remains in nucleus
- new genetic material not passed onto progeny ( genetic alteration not permanent)
- does not require selection
- DNA vectors or RNA can be used
- rapid process - cells can be harvested in 24-96 hours
- generally not suitable for studying vectors with inducible promotors

Stable:
- Transferred DNA integrates into genome
- genetic information is passed onto progeny (permanent alteration)
- requires selective screening for the isolation of stable transformants
- only DNA vectors can be used
- requires 2-3 weeks of selection for the isolation of stably transfected colonies (animal cells) or transformed plant cells germinated on selection
- suitable for the study of vectors with inducible promotors

Question 8

Stable transformation of animal cells

Answer 8

1) cells are transformed with a plasmid by either chemical or lipid treatment that contains a gene for drug resistance e.g. neomycin phosphotransferase (neo.) A negative control - a plasmid that does not contain the drug -resistance marker is included in the experimental plan

2) 48 hours after transformation the cells are diluted and plated onto medium containing the appropriate selection drug, G-418 is used for neoselection

3) over 14 days the drug containing medium is replaced every 3-4 days to keep up selection pressures

4) drug resistant cell clusters (clusters of transformed cells) appear in 2-5 weeks, depending on the cell type. Cell death occurs in 3-9 days in cells transformed with the negative control plasmid

5) transformed cell cultures are maintained in a medium containing the appropriate selection

6) stably transformed cells can be inserted into embryos and transplanted into females to develop into transgenic offspring

Question 9

Stable transformation for plants
(Agrobacterium infection aka floral dipping)

Answer 9

Soil bacterium Agrobacterium tumefaciens can infect plant cells to produce a tumour called a crown gall, formed of genetically mutated aberrant plant cells.
Agrobacterium contains a plasmid that contains T-DNA (transfer DNA) within the total plasmid which is called the Ti plasmid (tumour inducing plasmid)
The T-DNA can insert into the plant genome - it does this at random - it is a natural process.

Ti plasmid must be modified for use in plant cloning

A natural octopine Ti plasmid contains:
T-DNA for insertion
other genes to move T-DNA and to produce compounds that cause the plant cells to divide uncontrollably forming tumours

To create a Ti vector plasmid the natural Ti plasmid is modified to not produce tumours. The TDNA region is kept flanked by short repeats, in this region is added a Multiple cloning site and a plant resistance gene to be selected for in agrobacterium in addition to the naturally occurring antibiotic resistance gene on the original (natural) Ti plasmid.

The floral buds are then dipped into the solution of transformed agrobacterium (containing Ti vector plasmids) to induce stable transformation in the bud tissue and pollen.

Question 10

Transient transformation in plants (leaf infiltration)

Answer 10

These same transformed agrobacterium (containing Ti vector plasmids) are injected into the leaves of plants to induce transient expression.
The culture is pushed into the leaves with a syringe without a needle then the plants are left to grow for 2-3 days before assays or observations are carried out to check if the desired gene is being expressed.
e.g. under a fluorescence microscope you can observe a GFP reporter protein linked to say an actin protein - by observing that the cytoskeleton is now fluorescing you can confirm that the protein is present (actin proteins form the cytoskeleton)

Question 11

stable expression in plants - floral dipping summary

Answer 11

1) plants grown through mesh until they produce flower buds
2) turned upside down and dipped in agrobacterium culture
3) the agrobacterium infects the plant buds and T-DNA is transferred to the developing pollen
4) dipped plants are left to produce seeds (4-6 weeks)
5) seeds are harvested and planted on selective growth medium ( with an antibiotic e.g. with Karamycin)
6) seedlings that grow are resistant to the antibiotic (e.g. Karamycin) they are transplanted into soil and grown up to produce seeds - these plants are transgenic

Question 12

Working with transgenic organisms: aims

Answer 12

1) monitoring gene expression by reporter genes e.g. GFP or Luciferase allow visual monitoring
2) generating mutants: reverse genetics to learn about the functions of a specific gene- by knocking a gene out and observing any phenotypic changes that can help to identify the role of that gene

Question 13

monitoring: gene expression can be monitored in transient and stable transformants

Answer 13

this is done using reporter genes
A typical reporter gene expression vector has:
a gene or promotor inserted into the MCS which is linked to a reporter gene (e.g. GFP gene.) The reporter has its own terminator but no promotor so this is why you need to insert a promotor in the MCS. An antibiotic resistance gene should also be included so you can select for transformants

Question 14

reporter gene example: Beta galactosidase (Lac z)

Answer 14

Substrate: 5-bromo-4-chloro-3-inodyl-betaD-galactopyranoside (X-gal)
Product: insoluble blue dye ( non-quantitative)
used for blue/ white colony selection on petri dishes
useful for histology - dead samples fixed with ethanol

for colonies on agar:
a white colony consists of bacteria carrying a recombinant plasmid (Lac Z interrupted)

the blue dye can also be used as a marker in drosophila and mouse embryos.

How it works:
beta galactosidase is an enzyme that breaks down a substrate called X-gal to an insoluble blue dye, recombinant plasmids feature a gene that interrupts the Lac Z gene so that it cannot break down X-gal identifying these (white) colonies as transformant colonies.

Question 15

Reporter gene example: Beta glucuronidase (GUS)

Answer 15

Substrate: 5-bromo-4-chloro-3-inodyl-glucuronide (X-Gluc)
Product: insoluble blue dye
- non-quantitative, used for tissue staining/ histology

Substrate: 4-methylumbelliferyl-beta-D-glucoronide (MuG)
Product: fluorescent dye
- quantifiable, used in quantitative assays

( for both substrates the tissue must be fixed/ ground up)

Question 16

Reporter gene example: Luciferase

Answer 16

Substrate: luciferin
Product: light - quantitative by live imaging
- derived from fireflies
- spray plants with substrate and set up with a camera to monitor

Question 17

Reporter gene example: GFP (green fluorescent protein)

Answer 17

Substrate: none
Product: green fluorescence - non-quantitative live imaging
- derived from jellyfish
- as it is not an enzyme it does not require a substrate, you’re monitoring the fluorescence of the protein itself
- monitored by confocal (fluorescence) microscopes
- other colours are also available: yellow, cherry, blue etc.
allowing multiple different reporter genes to be linked to different promotors and visualised at the same time.

Question 18

Making a reporter gene - questions to ask

Answer 18

1) live imaging or histological analysis? GFP/luciferase or LacZ/GUS
2) Quantitative? Luciferase/GUS
3) Transcriptional or translational fusion?
- localisation to tissue/cell type only - trascriptional
-subcellular localisation/ observing molecular interactions - possible with translational fusion

Question 19

Transcriptional fusion

Answer 19

• gene to be studied has its own promotor, protein coding region and terminator
• isolate the promotor of the gene of interest and clone it upstream of your reporter gene (e.g. GFP)
• the terminator used can be from either the gene of interest or the reporter gene
• so the expression of the reporter gene is now under control of the promotor of your gene of interest -regulated by cell specific proteins and transcription factors
• so reporter (e.g. GFP) is only expressed where the gene of interest would have been expressed
  > remember gene of interest is not expressed we are only using its promotor
• so when this is transcribed the mRNA produced is just the reporter gene (GFP)
• so GFP will only be present in cells where the promotor was active
• GFP can be detected but its exact location in the cell is not known - it just collects in the cytoplasm
  ^ it can be located in specific tissue and cell types

Question 20

Translational fusion

Answer 20

• the whole gene is included, the promotor and coding region are linked to a reporter gene and termination sequence
• so when this is transcribed you get a fusion transcript - a fusion protein
• your protein of interest is fused/ partially linked to a reporter gene
• usually GFP is used as it is really small allowing the protein of interest linked to it to move around and interact with other proteins and localise to where it would normally be within the cell and perform its function (if its an enzyme) despite being linked to GFP
• so GFP fusion protein is only expressed in cell types where the promotor was active and is localised to its place of function within these cells
• so you can observe localisation within specific cell types at a subcellular level and sometimes observe molecular interactions

Question 21

Transcriptional fusion steps

Answer 21

1) promotor region isolated by:
a) PCR with appropriate RE sites included or
b) from a genomic library using a gene probe. The clone would be sequenced and often the promotor region would be amplified by pcr with appropriate RE sites

2) promotor fragments and vector digested with REs and promotor ligated into the plasmid (in the MCS region)

3) sequence the plasmid to confirm cloning reaction (orientation - directional cloning)

4) Transform/ transfect into an organism

5) screen for GFP ( or other reporter used)

Question 22

Translational fusion steps

Answer 22

1) The promotor and the coding region could be isolated together by either PCR or from a genomic library. However it is also quite common to isolate and clone them separately. this is because: a) the genomic fragment may be large
b) an open reading frame is needed from the gene ATG all the way to the reporter gene stop codon, this is easier to check with cDNA as it has no introns

2) So promotor region would be isolated as described before and coding region isolated by RT-PCR or from a cDNA library

3) The cloning steps would be the same - RE digestion and ligation, inserting promotor and coding region into MCS

4) Sequence to confirm cloning reaction(s) (orientation check)

5) Transform/transfect into an organism

6) Screen for GFP