MadSci Network: Genetics
Query:

Re: What did genetic engineers do to create

Date: Tue Jan 30 13:03:00 2001
Posted By: Paul Szauter, Staff, Mouse Genome Informatics, The Jackson Laboratory
Area of science: Genetics
ID: 979508320.Ge
Message:

The jellyfish gene used to fluorescently label transgenic animals is the GFP 
(Green fluorescent protein) gene from the jellyfish Aequorea victoria. The 
'fluorescent bunny' web page (which looks like a hoax) has this brief, 
accurate information on GFP:

'After green fluorescent protein (GFP) was first isolated from Aequorea 
victoria and used as a new reporter system (see: Chalfie, M., Tu, Y., 
Euskirchen, G., Ward, W., Prasher, D. (1994). Green Fluorescent Protein as a 
Marker for Gene Expression. Science 263, 802-805) it was modified in the 
laboratory to increase fluorescence. See: Heim, R., Cubitt, A. B. and Tsien, 
R.Y. (1995) Improved green fluorescence. Nature 373:663-664; and Heim, R., 
Tsien, R. Y. (1996). Engineering green fluorescent protein for improved 
brightness, longer wavelengths and fluorescence resonance energy transfer. 
Current Biology 6, 178-182. Further work altered the green fluorescent 
protein gene to conform to the favored codons of highly expressed human 
proteins and thus allowed improved expression in mammalian cells. See: Haas, 
J, Park, EC and Seed, B. (1996). Codon usage limitation in the expression of 
HIV-1 envelope glycoprotein. Current Biology 6: 315-24. More recently, new 
mutations with greater fluorescence have been developed. See: Yang, Te-Tuan 
et al. (1998). Improved fluorescence and dual color detection with enhanced 
blue and green variants of the green fluorescent protein. The Journal of 
biological chemistry, V. 273, N. 14, p. 8212. For a comprehensive overview 
of green fluorescent protein as a genetic marker, see: Chalfie, Martin. 
Kain, Steven. Green fluorescent protein : properties, applications, and 
protocols (New York : Wiley-Liss, 1998). Since its first introduction in 
molecular biology, GFP has been expressed in many organisms, including 
bacteria, yeast, slime mold, many plants, fruit flies, zebrafish, many 
mammalian cells, and even viruses. Moreover, many organelles, including the 
nucleus, mitochondria, plasma membrane, and cytoskeleton, have been marked 
with GFP.'

This is quoted from:
 http://www.ekac.org/
gfpbunny.html

By the way, I think that this site is a hoax because it was put up by a sort 
of performance artist and the picture of the green bunny is obviously fake. 
It wouldn't surprise me if there were some GFP rabbits (there are certainly 
transgenic rabbits). Sorry if this wasn't the story that you were referring 
to.

To explain GFP a little, proteins are polymers of amino acids. Twenty 
different amino acids are used in the synthesis of proteins. These amino 
acids differ in the chemical nature of their side chains, which can range 
from a simple hydrogen atom to a complex double-ring structure. Some of the 
amino acids are somewhat fluorescent, and depending on the exact sequence of 
the protein and the way the protein folds, some proteins are quite 
fluorescent. The GFP protein is a really striking example of a protein that 
is naturally fluorescent without having chemical dyes attached to it. The 
gene for GFP has been modified to make it more fluorescent, and to change 
the color of the fluorescence. Modified GFP genes are available 
commercially. See, for example:
 http://www.clontech.com/gfp/

There are pictures of GFP-expressing tissue culture cells, zebrafish, 
mosquitos and mice available in the Clontech GFP brochure, which you can 
view as a PDF file.

Some variant of GFP was used to make ANDi. The paper describing the creation 
of ANDi is:

Chan AW, Luetjens CM, Dominko T, Ramalho-Santos J, Simerly CR, Hewitson L, 
Schatten G Foreign DNA transmission by ICSI: injection of spermatozoa bound 
with exogenous DNA results in embryonic GFP expression and live rhesus 
monkey births. Molecular Human Reproduction 2000 6:26-33. Oregon Regional 
Primate Research Center, Departments of Obstetrics, Gynecology and Cell 
Developmental Biology, Oregon Health Sciences University, 505 NW 185th 
Avenue Beaverton, OR 97006, USA. 

These researchers used a technique called intracytoplasmic sperm injection. 
In this technique, DNA consisting of a retroviral vector containing the GFP 
gene was used to coat rhesus monkey sperm. The sperm was microinjected into 
rhesus monkey eggs to create embryos. The technique of intracytoplasmic 
sperm injection (ICSI) is used in human fertility clinics as a method of 
creating human embryos for implantation in women. In fertility clinics, of 
course, the sperm are not coated with foreign DNA.

When the DNA-coated rhesus monkey sperm was injected into the egg, it 
fertilized the egg to create a monkey embryo, but also allowed the 
retrovirus to infect the embryo cells and cause copies of its genetic 
material, including the GFP gene, to be inserted randomly into the monkey 
chromosomes. Earlier work by these researchers showed GFP expression 
(meaning synthesis of the GFP protein) in embryo cells. Some live births 
resulted from embryo implantation, but none of the monkeys carried the GFP 
gene in their chromosomes in the earlier work.

There are a number of techniques besides ICSI to generate transgenic 
animals. In mice, two general sorts of techniques are used. One is the 
injection of foreign DNA into one-cell mouse embryos. In this technique, 
mouse eggs are fertilized in vitro. A glass microneedle and micromanipulator 
apparatus is used to inject a small amount of a DNA solution into the 
nucleus of a one-cell embryo. The transgene DNA might be in a retroviral 
vector, which would allow a copy of the gene to transfer itself to the mouse 
chromosomes as the monkey transgene did. It can also be in a 'conventional' 
cloning vector like a bacterial plasmid; somehow, this sort of DNA can 
integrate into the mouse chromosomes using the same enzymes in the cell that 
repair damage to DNA. The problem with making transgenic mice (or monkeys) 
this way is that the DNA might integrate anywhere in the chromosomes, 
including in the middle of an important gene. Researchers are also not able 
to control the number of copies that integrate, and transgenic animals 
produced this way generally have multiple copies of the transgene. For many 
experiments, it wouyld be better to have a single copy at a known 
chromosomal location.

The other technique for making transgenic mice is called 'targeting'. This 
technique has not yet been extended to other mammals, but it could be. In 
this method, a special cell line derived from mice is used. The cell line 
(cells that can be grown in tissue culture) is called an embryonic stem cell 
(ES cell) line. These cells can be grown in a special medium in dishes in an 
incubator, but have the property that they can be incorporated into mouse 
embryos and differentiate into all kinds of mouse tissues, including the 
germ line (the cells that give rise to sperm and eggs). To make a chimeric 
embryo, a mouse embryo at the blastocyst stage is mixed with ES cells, and 
the aggregate is implanted into a foster mother. The ES cells are from a 
mouse strain that has light fur. The blastocyst is usually taken from a 
strain that has black fur. This way, when chimeric mice are born, they can 
be recognized by patches of black and light fur. These are bred to other 
mice, and if the ES cells are part of the germ line, the genes of the ES 
cells will be passed on to the progeny.

To make transgenics using this technique, the ES cells are transformed (that 
is, they take up DNA) using a very special DNA construct. DNA from the gene 
to be modified in the ES cells is incorporated into a DNA construct using 
recombinant DNA techniques. If you imagine the gene as having the sequence 
A-B-C, a construct is made that has the sequence A-C. This DNA is put into 
the ES cells, where it integrates into the mouse chromosomes by a process 
called recombination. This process uses enzymes that ordinarily repair DNA 
based on matching similar sequences using base pairing. The recombination 
event replaces one of the two chromosomal copies of the gene with the 
sequence A-B-C with the construct that has the sequence A-C. Since a piece 
of the gene is missing, the gene usually doesn't work anymore. The mice are 
OK because they have a second copy. When the progeny are inbred, some 
embryos are made that have two copies of the 'knock-out' (as it is called). 
These might die as embryos if the gene is important for development, or they 
might be born as mice that have a mutant phenotype. This technique is very 
useful, because the normal copy of the gene is replaced with a copy that has 
the sequence desired by the experimenter. Before the transformed ES cells 
are used to make mice, the experimenter verifies that the modified gene has 
replaced one of the chromosomal copies. Usually a gene making the cells 
resistant to a toxic drug is added to the construct. That way, it is 
possible to kill all the ES cells that didn't corporate the gene by adding 
the drug to the tissue culture medium.

Here is a site with a cartoon showing the creation of mouse knockouts:
 http://
www.ri.bbsrc.ac.uk/molbiol/mcwhir/embryoni.htm

I hope that this explanation is helpful to you. It is very hard to write 
about this subject without using genetics terms. We have an online glossary 
of such terms, with links to pictures, in our database. You can see our 
Glossary at:
 http://
www.informatics.jax.org//userdocs/glossary.shtml

Yours,

Paul Szauter
Mouse Genome Informatics



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