研習報告與討論
姓名：曾煥昌
學號：891632
研習地點：陽明大學神研所
Targeted expression of Cre recombinase to various brain regions
under doxycycline control in transgenic mice
1. Abstract
Functional analysis of mammalian gene relies on targeted mutations generated by
homologous recombination in mice. Many targeted inactivations of gene, however,
result in a pleiotropic phenotypes that are difficult to analyze. Conditional knockout
approach is desirable to direct tissue- or temporal- specific excision of the gene of
interest. Taking advantages of both the reverse tetracycline-inducible transactivator
(rtTA) and the site-specific Cre/loxP recombination systems, we designed a strategy to
generate transgenic mice bearing inducible Cre expression ability. In the core
construct, the reverse tetracycline transactivator (rtTA) is driven by
brain-specific ”nestin” promoter, and placed in cis regulation, the Cre recombinase is
controlled bt the minimal promoter containing tetracycline responsive operon.
2. Introduction
The key system of conditional knockout is Cre/loxP system. Cre is a 38 kDa
recombinase protein from bacteriophage P1, which mediates intramolecular (excisive
or inversional) and intermolecular (integrative) site specific recombination between
two loxP sites A loxP site consists of two 13 bp inverted repeats separated by an 8 bp
asymmetric spacer region.
One molecule of Cre binds per inverted repeat or two Cre molecules line up at one
loxP site. The recombination occurs in the asymmetric spacer region. Those 8 bases
are also responsible for the directionality of the site. Two loxP sequences in opposite
orientation to each other invert the intervening piece of DNA, and two sites in direct
orientation dictate excision of the intervening DNA between the sites leaving one loxP
site behind. This precise removal of DNA can be used to eliminate an endogenous
gene. Such a system is useful to investigate tissue-specific gene inactivation. In order
to excise target gene in a tissue-specific manner, two mouse lines are required. First, a
conventional transgenic mouse line with Cre targeted to a specific tissue or cell type,
and secondly a mouse strain that embodies a target gene to be flanked by two loxP
sites in the same direction ("floxed gene"). Recombination (excision and consequently
inactivation of the target gene) occurs only in those tissues (or organs) that expressing
Cre recombinase. Hence, the target gene remains active in all tissues that do not
express Cre recombinase.
fig.1
Another system adapted in this study is the rtTA/TRE system (and sometimes
called “Tet-on system” ). The tetracycline-responsive element (TRE) allows for the
expression of a transgene in the presence of tetracycline or its derivative, doxycycline.
When doxycycline is applied to the animal, it binds to the dimmerized transactivator
protein (rtTA), which in turn causes conformational change and allowing binding to
the tetracycline responding element (TRE). Transcription was controlled in such a
way that without doxycycline, no (or minimum) transgene will be produced.
fig.2
nn is the promoter of α2C4 receptor. This promoter expresses only in specific
tissue, such as some part of brain areas such as hippocampus and cerebellum.
3. Material& Methods
(1) Construct Plasmid
We must design a plasmid which contains recombinase and Tet-on system.
1. We select pSV-β –galactosidase (pSV-β –gal)plasmid for
2. our vector.
2. Sequentially Construct nn promoter, rtTA, Cre, TRE gene into the MCS of
pSV-β –gal plasmid. And design the trascription orientation to fit the following
figure.
fig.3
(2) Pronuclei Microinjection for Mice [4]
After constructing the plasmid, we should insert the DNA fragment into the
genome of mice. We use microinjection method to put the conditional knockout
system into mice.
1. To increase the yield and quality of eggs, female mice are superovulated with
gonadotrophins.
2.Fertilized embryos are harvested after dissection of the oviduct from newly plugged
mice.
3.Microinject the DNA fragment into the pronucleus of a fertilized ovum.
4. Reimplanted the microinjected embryos into the oviduct of pseudopregnant female
recipients, mated with appropriate timing to vasectomized male mice.
5. They will give birth 19-20 days after implantation.
(3) Analysis of Tail DNA for Transgene (southern boltting)
1. At 3-4 weeks of age, transgene integration can be assessed by tail tissue analysis.
2. Lyse the tail tissue and purify the genome DNA.
3. Digest the genome DNA with certain restriction enzyme and make the fragment run
in agarose gel by electrophoresis.
4. Transfer the DNA onto a special filter.
5. Use the previous construct DNA as probe and make it radioactive.
6. Hybrid the fragment of genome with probe.
7. Analyse radioactivity of the filter. Check the hybrid DNA size and intensity to
screen the transgenic mice.
(4) Mating The Transgenic Mice
At around 6 weeks of age, matings should be set up between transgenic mice.
Usually, the transgenic mice are heterozygotes. We will mate two heterozygotes to get
homozygotes. Then, we should do southern blotting experiment to the descendants to
screen homozygotes. Furthermore, in order to confirm the hetrozygotes, we do
testcross for the possible homozygote. In this way we can get a homozygote with our
construct system.
4. Result & discussion
In my summer research, I participated only the southern blotting experiment of the
series. During the two mouths, I screened about 120 mice and found thirty
heterozygotes.
As soon as I found these heterozygotes, we made them mate each other. But these
mice were too little to be analyzed their tails’ DNA so I did not find any homozygotes.
After a series of experiment, we can get transgenic mice with conditional knockout
system. As fig.1 show, we only establish half of the complete system. When we want
to to investigate some interesting gene, we just use the knockin method to put loxP
sequence on both sides of the gene. Then, mate the two kinds of transgenic mice. We
can obtain a whole conditional knockout system. Furthermore, if we change the gene
between two loxP sequences we can study variable gene in the same way.
This experiment was my first job in my summer lab. Although I can’t finish the
whole experiment, I have learned how the research acts. Besides the Southern boltting,
I learned ICC (immuno-cytochemistry), which can locate specific protein expression
by antibody. It was not my job but I asked to teach me . Therefore it wasn’t a
complete plan and I didn’t describe above.
This research experience let me find interests of experiment and sometimes feel
frustrated. It is important to find out what is the mistake when I get no expected result.
Although the life of research is not easy, I hope that I can find out some significant
result to change the life of humans in whole world.