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OK.  So last time I reviewed for you
some of the basic cloning techniques.

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I failed to show you this slide,
which I intended to do, which is a

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commercially available plasmid.
I told you about plasmids last time,

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small circular DNA molecules that
are used in the purpose of cloning.

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They're derived from nature, but
then they've been heavily

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manipulated by scientists to be
useful for the purposes of cloning.

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Recall that they have two,
three critical elements, an origin

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of replication to allow them to be
replicated inside of bacteria,

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a selectable marker, a
drug-resistance gene,

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ampicillin resistance gene for
example, and finally,

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can you now show the middle slide?
He's working on it.  And finally

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what we call a multiple cloning site,
a set of restriction sites,

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restriction enzyme recognition sites
that allow us to pop

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in pieces of DNA.
I have dual paper readers.

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Dual paper readers?  That doesn't
happen very often.

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You usually have one but not often
two in a row.  Good.

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So these are useful practical tools.
Maybe before I forget,

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I'll mention, remind you of the quiz
on Monday, where you go based on

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your last name.
There was a discussion last time

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about the review session,
which is actually not on here but we

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didn't change the review session.
Oh, because it was last night.

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We weren't able to change the review
session, which was last night,

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but there is a tutoring session
which will cover the same material

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from today between 4:00 and 6:00.
And then there are additional

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sessions with the TAs,
including up till Monday morning

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before the exam.
So there should be ample

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opportunity to get your questions
answered.  So plasmids are now sort

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of a useful and are a
very available tool.

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This figure comes from your book.
It's a version of what we covered

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in our example last time,
the cloning of the T gene.

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Here in red is the starting
material, the DNA from the source.

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In our case it was S. pyogenes,
that flesh eating bacterium,

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but it could be anything.  It could
be genomic DNA from you.

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You take that genomic DNA,
whatever the source material is,

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you digest it, cut it with a
restriction enzyme,

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with a particular restriction enzyme.
You then mix that DNA with plasmid

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DNA which has been cut with the same
restriction enzyme,

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the sticky ends of the linear
molecule anneal with the sticky ends

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of the plasmid.
You then add DNA ligase to seal

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those nicks to produce full
covalently closed circular molecules

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that are composed of both the
plasmid and now an insert

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from the DNA sample.
You then take those recombinant

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plasmids, mix them with bacteria
under conditions which allow the DNA

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to get into those bacterial cells.
This is a process called

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transformation.
You then plate those bacteria onto

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auger plates that contain the
antibiotic, in this case ampicillin

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in our example.
The bacteria that didn't get a

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plasmid die.  The bacteria that did
get a plasmid survive and form

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colonies on the dish.
And then the question I left you

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with was how are we going to find
the colony or colonies that carry

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the T gene?  How are we going to
find the gene,

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the recombinant plasmid,
the colony carrying the recombinant

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plasmid with the T gene?
I also want to mention a bit of

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terminology, which we will come back
to later.  We often refer to the

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collection of colonies,
the collection of recombinant

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plasmids contained within those
bacteria as a library.

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Like a library containing books,
this is a collection of different

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things.  In this case,
different recombinant plasmids which

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you can go back to repeatedly taking
isolates from the colony.

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In our case we would then grow up
the individual bacteria to get ample

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amounts of the plasmid of interest.
And this term library is used in

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molecular biology a lot,
a diverse collection of clone

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fragments to which you can return
depending on your specific needs.

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So how are we going to isolate our
plasmid of interest?

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Well, if you recall,
we knew the sequence of the genome

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of S. pyogenes.
And, therefore,

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we knew the sequence of the T gene.
So imagine that the T gene had a

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particular sequence starting from
the 5 prime end of

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A-G-G-C-T-G-G-T-G-G-G-A
to the 3 prime end.

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So this is imbedded within the T
gene.  The reverse complement on the

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other strand reading in this
direction from the 5 prime end would

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be T, did I say that,
what's this?  C-C-C-A-C-C-A-G-C-C-T

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3 prime.  OK?  So we know a bit
about the thing we're

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interested in.
We know its sequence.

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And we can use this information to
our advantage to isolate the plasmid

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that carries this fragment.
So the bacteria, as shown on this

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slide, are present in colonies.
Each colony contains a recombinant

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plasmid.  The first step in
isolating the plasmid of interest is

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to make a copy of the plate that
carries these bacteria to replicate

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what's on this plate onto a piece of
filter paper.

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We call this replica plating.

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And in this case we're going to do

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it onto a filter,
a piece of paper or nylon to make a

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copy of the colonies and the pattern
of the colonies onto something else

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that we can manipulate.
So we make an exact copy.

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And this done by literally placing
the piece of filter paper onto the

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Petri dish, as shown here.
Here is the Petri dish with the

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colonies.
You place a filter on top of those

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colonies.  They'll stick to it.
Some of the cells in those colonies

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will stick to the filter.
You then pull the filter off and

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you get some of the colonies,
some of the cells sticking in

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exactly the place where they were on
the original plate.

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You can actually place this on
another plate and allow those cells

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to grow directly on the piece of
filter paper.  So you make an exact

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copy of what was here onto a piece
of paper.  Now,

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what are you going to do with that?
Well, we're going to use this

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information that we have about the
sequence of the gene to isolate the

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colony of interest.
First thing we do is to lyse the

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bacteria that are now present on
this filter and denature the DNA.

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What I mean by denature the DNA is

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to pull it apart.
It's double-stranded when it's

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present in the bacteria.
To denature DNA is to make it

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single-stranded.
And typically the way we do that in

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these applications is to place a
piece of filter paper under high pH

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conditions, and it causes the DNA
strands to melt apart.

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So now the plasmids, which were
double-stranded in these bacterial

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cells, were double-stranded here
have now been pulled apart into

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single-stranded molecules.
So, on this piece of filter paper

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there are cells with unwound DNA,
single-stranded DNA in them in this

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same pattern, which I won't try to
reproduce here.

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And the question is how are we
going to identify which colony

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carries a piece of the T gene?
How are we going to do that?  Does

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anybody have a clue?
All you need to know is present up

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here.  Yeah?

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So you're thinking of the example
from the book in which the,

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in the example in the book they used
a plasmid that had two selectable

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markers.  And you were interested in
those that grew under one antibiotic

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selection and didn't grow under a
different antibiotic selection.

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In our case, in our plasmid, we
actually didn't have two selectable

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markers.  We only had one.
So we're not going to use that

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strategy.  We have to rely on some
of the information I've

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given you already.
Well, we know the sequence of the T

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gene.  We know,
for example, this little bit of the

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T gene, so we can make some that we
call a probe.  A probe,

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for this application, is an
oligonucleotide,

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a small segment of nucleotides that
we can synthesize.

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You can actually make any DNA
sequence you want using

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chemical synthesis.
This has now been fully automated.

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So you can just punch the sequence
into a computer,

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attach it to one of these machines,
the machine will go off and make a

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linear piece of DNA of any sequence
you want.  It's like a typewriter

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almost.  You type it in,
and what you get back out is a piece

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of DNA that is the sequence that you
typed in.  So we can make any DNA we

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want such that we could make a piece
of DNA that had the sequence

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A-G-G-C-T-G-G-T-G-G-G-A.

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OK?  We could make that piece of DNA.
Moreover, we could modify that

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piece of DNA with enzymes to add a
radioactive phosphate at this 5

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prime end.  So we could make a radio
labeled probe.

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So now that we have that,
what are we going to do?  Anybody?

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Yes.  Yes.

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Right.

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Exactly.  So this is now
complementary to the other strand.

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The DNA within these cells that
have been lysed and now stuck onto

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this filter are also single-stranded.
So this complementary strand is

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going to be sitting in a single
stranded conformation ready to

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anneal, hybridize with this probe.
So you apply this radioactive probe

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to this filter.
It will bind somewhat

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indiscriminately at first,
but it will bind very tightly to

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that complementaryary strand from
the T gene.  And remember there are

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probably a million bacterial cells
within this colony,

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so there will be a million copies of
the probe anneal to this colony.

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You do have to do a little washing
to get rid of the nonspecific

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binding which will take place.
So you wash the cells, or wash the

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filter, get rid of the
nonspecific binding.

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The specific binding will withstand
the wash.  And then you apply a

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piece of x-ray film.
And what you'll end up with is a

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faint image of the filter,
some very faint signal from the

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colonies that don't hybridize,
and a very strong signal from the

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colonies that did hybridize properly
to the probe.  OK?

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So now you know, based on the
position on that filter,

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which colony it is that carries the
T gene.  And in this case it's that

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one right there.
So we can go back to that master

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plate that we've kept,
use a toothpick, pluck off that

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colony and grow it up into high
concentrations successfully having

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completed our cloning project of the
ever toxic T gene.

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And that's gone through here in
this figure, if you want to look at

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it again.  You take this filter that
has the colonies.

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It's actually the step here where
you grow the cells up on

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the filter itself.
You then lyse the cells,

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denature the DNA, add the
radioactive probe.

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It won't hybridize strongly to
those colonies that don't carry the

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clone of interest.
It will hybridize strongly to the

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colonies that do have the clone of
interest.  And then through this

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x-ray film method you can visualize
where those colonies are and then go

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back and pick the colonies from the
master plate.  OK?

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So that's how it works.
It's fairly straightforward and

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works extremely well to give you the
cells and clones of

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interest.  Woops.

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So that's what we call clone
identification by hybridization

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using a radioactive probe.
And I should mention, because it

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will come up again later,
that we use these radioactive probes

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extensively for molecular biology.
And we use non-radioactive probes,

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oligonucleotide synthesized in the
same way that I mentioned for all

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sorts of molecular biology
applications in this era.

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And you'll see examples later in
the lecture.  I want to briefly

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mention another technique known as
cloning by complementation.

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In this instance, we're going to
clone by the function of the plasmid

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that we've introduced
into the cells.

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Not its sequence.
By its function.  And the example

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I'm going to give you is to clone a
gene that you might know about based

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on the biochemistry of the organism,
but you haven't identified the gene

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that encodes that enzyme.
The example I'm going to give you

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is to clone an enzyme which is
responsible for catalyzing the

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breakdown of lactose,
a disaccharide.  And this is

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accomplished by an enzyme called
beta-galactosidase.

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And it converts glucose.
It converts lactose to glucose and

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galactose.  And this is an important
enzyme in bacteria and also in you.

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The enzyme is encoded by the lacZ
gene of bacteria.

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OK?  And this exercise that we're
going to go through is to try to

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isolate the lacZ gene through this
method called cloning by

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complementation.
We need to use a trick,

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which I'll show you on this slide.
And that is that there's an

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artificial substrate for this enzyme
which is called in the field X-gal.

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It's related to lactose.  The
important thing is that when X-gal

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gets cleaved by beta-galactosidase,
it releases galactose and this

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molecule which precipitates and
turns blue.  So we have a visual

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indicator of beta-galactosidase
activity by placing in the medium,

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either in liquid medium or on a
Petri dish, this substance X-gal.

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We know whether or not the cells
have beta-galactosidase activity by

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virtue of whether the cells or the
colonies turn blue.

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OK.  So the first thing that we're
going to do in this technique is to

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take some wild type E. coli.

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If we take a liquid culture of wild
type E. coli and we plate them onto

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a tissue culture,
a Petri dish that contains X-gal,

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00:19:47,000 --> 00:19:52,000
this substrate that in the presence
of beta-galactosidase will turn blue,

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will get colonies.
And what color will those colonies

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be?  Blue.
These are normal E.

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coli.  So they're able to metabolize
the X-gal and produce this blue

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pigment.  OK?  Now,
I'm interested in the enzyme,

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the gene that encodes the enzyme
that carries out this activity.

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00:20:17,000 --> 00:20:22,000
And the way that I'm going to get
to it is first to isolate mutant E.

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00:20:22,000 --> 00:20:27,000
coli.  It cannot do this reaction.
I'm going to try to isolate a mutant

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00:20:27,000 --> 00:20:32,000
strain that's deficient in
beta-galactosidase activity.

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00:20:32,000 --> 00:20:37,000
And the way that I do that is to
mutagenize the wild type E.

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00:20:37,000 --> 00:20:42,000
coli.  I can do this by physical
methods or more commonly by just

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00:20:42,000 --> 00:20:48,000
adding a chemical mutagen to the
broth that the E.

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00:20:48,000 --> 00:20:53,000
coli is growing in.
I mutagenize the cells and then I

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00:20:53,000 --> 00:20:59,000
plate them onto a plate
that has X-gal.

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00:20:59,000 --> 00:21:03,000
Now, most of the cells that got
mutagenized will have mutated some

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00:21:03,000 --> 00:21:08,000
other gene, not the
beta-galactosidase gene,

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00:21:08,000 --> 00:21:13,000
some other gene, or maybe no gene,
such that what color will those

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00:21:13,000 --> 00:21:18,000
colonies be?  They'll be blue
because they still have a functional

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00:21:18,000 --> 00:21:23,000
beta-galactosidase enzyme.
They still have a wild type lacZ

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00:21:23,000 --> 00:21:28,000
gene.  So most of the colonies will
be blue.  But what if the cell

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00:21:28,000 --> 00:21:33,000
incurred a mutation in the lacZ gene
that knocked out lacZ function?

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00:21:33,000 --> 00:21:36,000
What color will the colony be?
Not blue.  It will be white.  OK?

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00:21:36,000 --> 00:21:40,000
And we're going to assume, for the
sake of argument,

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00:21:40,000 --> 00:21:44,000
that if you get a white colony it
means that the cells have a mutation

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00:21:44,000 --> 00:21:48,000
in this gene lacZ.
That's actually not a safe

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00:21:48,000 --> 00:21:52,000
assumption.  It could have a
mutation in some other gene,

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00:21:52,000 --> 00:21:56,000
but we're not going to worry about
that today.  Just say that if we get

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a white colony we're going to assume
that the cells have a mutation

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00:22:00,000 --> 00:22:06,000
in the lacZ gene.
So now we want to clone the lacZ

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00:22:06,000 --> 00:22:15,000
gene.  Our goal is to clone the lacZ
gene.  What are we going to do?

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00:22:15,000 --> 00:22:25,000
What are we going to do?  Well,
I'll give you the first clue.  We're

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00:22:25,000 --> 00:22:33,000
going to make a library.
We're going to make a library of

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00:22:33,000 --> 00:22:39,000
fragments of DNA some of which will
carry the lacZ gene.

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00:22:39,000 --> 00:22:45,000
From what are we going to make that
library?  What will be our source

249
00:22:45,000 --> 00:22:51,000
DNA to make that library?
Will it be these cells isolated

250
00:22:51,000 --> 00:22:57,000
from this colony or will it be some
other cell?  Do these cells have a

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00:22:57,000 --> 00:23:02,000
functional lacZ gene?
No.  Do these cells?

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00:23:02,000 --> 00:23:08,000
Yes.  So you want to make a library
from wild type E.

253
00:23:08,000 --> 00:23:13,000
coli.  So that you have a bunch of
plasmids that carry fragments of the

254
00:23:13,000 --> 00:23:19,000
E. coli genome some of which carry
the lacZ gene.

255
00:23:19,000 --> 00:23:25,000
What am I going to do with that
library?  What am I going to do with

256
00:23:25,000 --> 00:23:39,000
that collection of clones?

257
00:23:39,000 --> 00:23:45,000
I'm going to transform it en masse
all the different clones to a

258
00:23:45,000 --> 00:23:51,000
population of cells.
What cells will I transform it into?

259
00:23:51,000 --> 00:23:57,000
Which bacteria?
Would I put those ones into the

260
00:23:57,000 --> 00:24:05,000
lacZ mutants?

261
00:24:05,000 --> 00:24:10,000
And then I plate that transformation
onto plates that contain amp.

262
00:24:10,000 --> 00:24:16,000
I need the drug to identify those
cells that have picked up any

263
00:24:16,000 --> 00:24:21,000
plasmid because I don't care about
cells that haven't picked up any

264
00:24:21,000 --> 00:24:27,000
plasmid so I use amp plates.
And what else do the plates have in

265
00:24:27,000 --> 00:24:32,000
them?
The indicator of lacZ activity,

266
00:24:32,000 --> 00:24:37,000
X-gal.  So I'm going to take these
transformants,

267
00:24:37,000 --> 00:24:42,000
which were derived from these cells,
and I'm going to plate them out onto

268
00:24:42,000 --> 00:24:46,000
a plate.  What color will most of
the colonies be?

269
00:24:46,000 --> 00:24:51,000
Will most of them,
raise your hand.  Will most of them

270
00:24:51,000 --> 00:24:56,000
be white?  Will most of them be blue?
In fact, most of them will be white

271
00:24:56,000 --> 00:25:01,000
because most of the cells did not
pick up a plasmid that carries the

272
00:25:01,000 --> 00:25:06,000
functional lacZ gene.
Most of the cells picked up a

273
00:25:06,000 --> 00:25:10,000
plasmid that carried some other
piece of the E.

274
00:25:10,000 --> 00:25:15,000
coli genome.  OK?
So most of the cells will not be,

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00:25:15,000 --> 00:25:20,000
will not have functional lacZ
activity, they'll be white.

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00:25:20,000 --> 00:25:24,000
However, at some frequency a clone,
a cell will pick up a recombinant

277
00:25:24,000 --> 00:25:29,000
plasmid that does carry the lacZ
gene and that colony

278
00:25:29,000 --> 00:25:34,000
will turn blue.
And that's the colony that we're now

279
00:25:34,000 --> 00:25:38,000
interested in because it has
reconstituted lacZ activity.

280
00:25:38,000 --> 00:25:42,000
The mutant, the recombinant DNA has
complemented the mutation.

281
00:25:42,000 --> 00:25:51,000
So I could isolate this plasmid and

282
00:25:51,000 --> 00:25:54,000
sequence it.  And based on the
sequence I might have confidence

283
00:25:54,000 --> 00:25:57,000
that indeed it is the lacZ gene.
Remember I told you that there was

284
00:25:57,000 --> 00:26:00,000
an assumption built in here that
this mutation really did affect the

285
00:26:00,000 --> 00:26:04,000
lacZ gene?  And that's why the cells
were white.

286
00:26:04,000 --> 00:26:07,000
That was an assumption.
And to confirm that assumption we

287
00:26:07,000 --> 00:26:10,000
could look at the sequence of the
clone that we isolated and figure it

288
00:26:10,000 --> 00:26:14,000
out, figure out whether it's the
right enzyme.  OK?

289
00:26:14,000 --> 00:26:17,000
So I find that teaching this is
always a little confusing.

290
00:26:17,000 --> 00:26:20,000
I tried to simplify it today and go
through it slowly.

291
00:26:20,000 --> 00:26:24,000
Hopefully you took notes and you
can think about it on your own.

292
00:26:24,000 --> 00:26:27,000
It is relatively straightforward
and it's a useful concept to

293
00:26:27,000 --> 00:26:30,000
understand that A) you could isolate
mutations, and B) you can find the

294
00:26:30,000 --> 00:26:34,000
genes that were mutated by
complementing those mutations

295
00:26:34,000 --> 00:26:44,000
through cloning.  OK?

296
00:26:44,000 --> 00:26:47,000
OK.  So now that I've spent a
lecture and a half talking about

297
00:26:47,000 --> 00:26:51,000
cloning, I'm now going to tell you
that cloning, while it was

298
00:26:51,000 --> 00:26:54,000
incredibly important and still is,
has almost been superceded by

299
00:26:54,000 --> 00:26:58,000
another technique.
It's not that we don't use cloning.

300
00:26:58,000 --> 00:27:02,000
We actually use it all the time.
But it's been joined by another

301
00:27:02,000 --> 00:27:08,000
technique known as PCR which stands
for the polymerase chain reaction.

302
00:27:08,000 --> 00:27:14,000
And any of you who have worked in

303
00:27:14,000 --> 00:27:18,000
molecular biology labs have been
exposed to this because it is a

304
00:27:18,000 --> 00:27:22,000
totally pervasive technology.
Everybody uses it.  And it's not

305
00:27:22,000 --> 00:27:26,000
just in molecular biology labs.
Forensic labs.  Archeology labs.

306
00:27:26,000 --> 00:27:30,000
Anybody who's interested in
generating large amounts of DNA from

307
00:27:30,000 --> 00:27:35,000
a small amount of DNA uses this
technique called PCR.

308
00:27:35,000 --> 00:27:38,000
It was invented only a few years ago,
but it's incredibly important.

309
00:27:38,000 --> 00:27:41,000
In fact, the guy who invented it,
who was not a very well known

310
00:27:41,000 --> 00:27:45,000
scientist, won the Nobel Prize just
a couple of years after he invented

311
00:27:45,000 --> 00:27:48,000
it because it became so powerful so
quickly.  It's a very,

312
00:27:48,000 --> 00:27:52,000
very simple technique.  It's one of
these ah-ha techniques that after

313
00:27:52,000 --> 00:27:55,000
it's been invented and described
everybody says,

314
00:27:55,000 --> 00:27:59,000
oh, I could have thought of that.
But, actually, nobody did until

315
00:27:59,000 --> 00:28:02,000
this guy, Kary Mullis.
So I'm going to show it to you

316
00:28:02,000 --> 00:28:05,000
briefly on the board.
And then we're going to go through

317
00:28:05,000 --> 00:28:08,000
a movie that shows it again.
So hopefully you'll get how it

318
00:28:08,000 --> 00:28:12,000
works.  Importantly,
it relies on having some information

319
00:28:12,000 --> 00:28:15,000
about the DNA that you want to
amplify up in large quantities.

320
00:28:15,000 --> 00:28:18,000
Again, the goal here is to amplify
up a piece of DNA of interest to

321
00:28:18,000 --> 00:28:21,000
large quantities.
This technique is so powerful that

322
00:28:21,000 --> 00:28:24,000
you can use a single DNA molecule to
start.  You can use the amount of

323
00:28:24,000 --> 00:28:27,000
DNA that you get from hair from a
crime scene, for example.

324
00:28:27,000 --> 00:28:31,000
And that's mostly the way,
you know.

325
00:28:31,000 --> 00:28:34,000
What is it?  CSI Miami and stuff.
They use this technique all the

326
00:28:34,000 --> 00:28:38,000
time.  So you can use minuscule
amounts of DNA.

327
00:28:38,000 --> 00:28:42,000
Even a single molecule is enough.
But you do need to know a little

328
00:28:42,000 --> 00:28:51,000
bit of known sequence.

329
00:28:51,000 --> 00:28:54,000
So if you know a little bit of known
sequence you can use PCR.

330
00:28:54,000 --> 00:28:58,000
So the first thing you do is to
denature the DNA.

331
00:28:58,000 --> 00:29:05,000
And in this case we do that by heat.

332
00:29:05,000 --> 00:29:10,000
If you heat up a DNA molecule it
will also separate from itself.

333
00:29:10,000 --> 00:29:16,000
And we typically heat it up to
about 94 degrees.

334
00:29:16,000 --> 00:29:21,000
And the consequence of this is this
double-stranded piece of DNA

335
00:29:21,000 --> 00:29:26,000
molecule will separate into two
single-stranded pieces of DNA.

336
00:29:26,000 --> 00:29:32,000
And at that point we anneal,
hybridize onto the DNA --

337
00:29:32,000 --> 00:29:37,000
I'm not about to do another
demonstration here.

338
00:29:37,000 --> 00:29:43,000
We anneal onto the DNA
oligonucleotides,

339
00:29:43,000 --> 00:29:48,000
synthetic little fragments of DNA
that are complementary to this known

340
00:29:48,000 --> 00:29:54,000
sequence at this end and at this end.
So I synthesize a short

341
00:29:54,000 --> 00:30:00,000
oligonucleotide which will anneal to
this strand at this position.  OK?

342
00:30:00,000 --> 00:30:03,000
I should have written up the
polarities because people often get

343
00:30:03,000 --> 00:30:07,000
confused.  Here's the 5 prime end of
this DNA molecule,

344
00:30:07,000 --> 00:30:11,000
the 3 prime end of that DNA molecule,
the 3 prime end of that one,

345
00:30:11,000 --> 00:30:15,000
5 prime end of that one.  So if I'm
thinking about the top strand here,

346
00:30:15,000 --> 00:30:18,000
the 5 prime end here, the 3 prime
end here, and that little

347
00:30:18,000 --> 00:30:22,000
oligonucleotide,
which in this context we call a

348
00:30:22,000 --> 00:30:26,000
primer, would have its 5 prime end
here and its 3 prime end here.

349
00:30:26,000 --> 00:30:30,000
Thus, the arrow because this is the
direction of DNA synthesis.

350
00:30:30,000 --> 00:30:34,000
If we're going to synthesize DNA off
of this piece of DNA it's going to

351
00:30:34,000 --> 00:30:38,000
go in that direction.
And then we likewise order up and

352
00:30:38,000 --> 00:30:42,000
anneal onto an oligonucleotide that
binds to this strand of this

353
00:30:42,000 --> 00:30:47,000
sequence.  And,
again, to show the polarities,

354
00:30:47,000 --> 00:30:51,000
here's the 5 prime end.  Ah, thank
you very much.

355
00:30:51,000 --> 00:30:55,000
Here's the 5 prime end,
here's the 3 prime end, so that the

356
00:30:55,000 --> 00:30:59,000
oligonucleotide primer has its 5
prime end here, its 3

357
00:30:59,000 --> 00:31:13,000
prime end here.  OK.

358
00:31:13,000 --> 00:31:18,000
And now these primers are sitting in
such a way, annealed to a template

359
00:31:18,000 --> 00:31:23,000
piece of DNA with a 3 prime end
facing in this direction,

360
00:31:23,000 --> 00:31:29,000
that are ready to be extended by DNA
polymerase.

361
00:31:29,000 --> 00:31:37,000
Sorry.  I'm getting confused about

362
00:31:37,000 --> 00:31:43,000
my colors here.
You can't see them on the board?

363
00:31:43,000 --> 00:31:49,000
You can't see the blue.  OK.  All
right.  So the blue,

364
00:31:49,000 --> 00:31:55,000
sorry about this.  The blue will now
be orange.  OK?

365
00:31:55,000 --> 00:32:01,000
And this one will be pink.
OK?  So I'm going to extend this

366
00:32:01,000 --> 00:32:07,000
pink oligonucleotide with a
polymerization reaction to

367
00:32:07,000 --> 00:32:13,000
fill in this strand.
And I'm going to extend the orange

368
00:32:13,000 --> 00:32:19,000
guy likewise to fill in this strand.
OK?  So this is accomplished by

369
00:32:19,000 --> 00:32:26,000
addition of DNA polymerase and the
nucleotide precursors that are

370
00:32:26,000 --> 00:32:33,000
needed for DNA synthesis,
which we abbreviate dNTPs.

371
00:32:33,000 --> 00:32:37,000
And if I incubate that in that way
I'll get extension from this primer

372
00:32:37,000 --> 00:32:42,000
in this direction,
extension from this primer in this

373
00:32:42,000 --> 00:32:46,000
direction.  And what I've done here
is to go from one DNA molecule to

374
00:32:46,000 --> 00:32:51,000
two DNA molecules.
I've duplicated this piece of DNA

375
00:32:51,000 --> 00:32:56,000
in a test-tube.
The beauty of PCR is that you then

376
00:32:56,000 --> 00:33:01,000
go through this process again and
again and again and again.

377
00:33:01,000 --> 00:33:05,000
And each time you get an exponential
increase in the amount of DNA.

378
00:33:05,000 --> 00:33:09,000
You go from one to two to four to
eight and so on and so on and so on

379
00:33:09,000 --> 00:33:13,000
and so on.  And in a relatively
short time you can now have millions,

380
00:33:13,000 --> 00:33:18,000
billions or more copies of your DNA.
OK?  So that's the basic principle.

381
00:33:18,000 --> 00:33:22,000
And, again, we'll go through it in
a little more detail in the movie.

382
00:33:22,000 --> 00:33:26,000
As I said, the technique was
invented by Kary Mullis,

383
00:33:26,000 --> 00:33:31,000
an investigator at a company called
Cetus.

384
00:33:31,000 --> 00:33:51,000
Kary Mullis is an interesting
character.  I don't have time to

385
00:33:51,000 --> 00:34:11,000
tell you the full stories of Kary
Mullis.  As I told you,

386
00:34:11,000 --> 00:34:31,000
he won the Nobel Prize.  He's a real
California kind of a surfer dude.

387
00:34:31,000 --> 00:35:05,000
Very laid back.

388
00:35:05,000 --> 00:35:09,000
So, anyway, you can use this
technique for lots of applications,

389
00:35:09,000 --> 00:35:14,000
as shown here.  It's been
commercialized in many ways.

390
00:35:14,000 --> 00:35:18,000
We now have machines that will go
through these various stages of

391
00:35:18,000 --> 00:35:23,000
denaturation, annealing and
synthesis using a temperature

392
00:35:23,000 --> 00:35:28,000
regulated block that will go from 94
degrees to 68 degrees to 72 degrees

393
00:35:28,000 --> 00:35:32,000
to allow denaturation,
annealing and polymerization around

394
00:35:32,000 --> 00:35:36,000
this circle many, many, many times.
And if you do this,

395
00:35:36,000 --> 00:35:39,000
for example, 30 times,
if you go through this cycle 30

396
00:35:39,000 --> 00:35:43,000
times you make two to the thirtieth
copies of your DNA,

397
00:35:43,000 --> 00:35:46,000
which is ten to the ninth DNA
molecules, which is a ton.

398
00:35:46,000 --> 00:35:49,000
And that can be done in just a
couple of hours using a machine such

399
00:35:49,000 --> 00:35:53,000
as this.  OK.  So let me take you
through this movie.

400
00:35:53,000 --> 00:35:56,000
I'm going to cut it short because
the process is gone through

401
00:35:56,000 --> 00:35:59,000
in great detail.
The narrator is actually Paul

402
00:35:59,000 --> 00:36:03,000
Matsudaira who is a professor here
at MIT.  And Paul really sort of

403
00:36:03,000 --> 00:37:38,000
drags it on, but I'm going to --

404
00:37:38,000 --> 00:37:41,000
Basically all he says is what I said,
which is you take an individual DNA

405
00:37:41,000 --> 00:37:45,000
molecule for which you know
sequences, and you run through this

406
00:37:45,000 --> 00:37:48,000
reaction multiple times.
And each time you do you get a

407
00:37:48,000 --> 00:37:52,000
duplication of the DNA sequence.
And your book goes through it, too.

408
00:37:52,000 --> 00:37:56,000
So I will get this thing posted on
the Web.  It's not on Monday's quiz

409
00:37:56,000 --> 00:38:00,000
so you don't have to
worry about that.

410
00:38:00,000 --> 00:38:04,000
We can learn about it in the future.
Maybe I'll fix it so that on

411
00:38:04,000 --> 00:38:09,000
Monday's lecture or Wednesday's
lecture, Professor Sive who is

412
00:38:09,000 --> 00:38:13,000
giving that lecture can go through
it with you.  So sorry about this.

413
00:38:13,000 --> 00:38:18,000
I also wanted to mention briefly
that the enzyme that we use to carry

414
00:38:18,000 --> 00:38:23,000
out the polymerization,
the particular DNA polymerase that

415
00:38:23,000 --> 00:38:27,000
we use is isolated from what we call
a thermophilic bacterium isolated

416
00:38:27,000 --> 00:38:31,000
from the hot springs.
And that's important because we

417
00:38:31,000 --> 00:38:34,000
carry out this reaction at a very
high temperature.

418
00:38:34,000 --> 00:38:37,000
You'll notice that the
polymerization was done at 72

419
00:38:37,000 --> 00:38:41,000
degrees.  Now,
your DNA polymerases won't work at

420
00:38:41,000 --> 00:38:44,000
72 degrees but this organism grows
at very, very high temperatures so

421
00:38:44,000 --> 00:38:47,000
its DNA polymerase can function
there.  And this,

422
00:38:47,000 --> 00:38:50,000
again, has turned into a cottage
industry.  There are hundreds of

423
00:38:50,000 --> 00:38:53,000
millions of dollars worth of this
particular polymerase called Taq

424
00:38:53,000 --> 00:38:57,000
polymerase sold for this
application.  OK.

425
00:38:57,000 --> 00:39:02,000
So let's carry onto the next topic
then.  So what we've been talking

426
00:39:02,000 --> 00:39:07,000
about now is transferring DNA in the
case of cloning or amplifying DNA

427
00:39:07,000 --> 00:39:12,000
for various applications.
For some applications that's not

428
00:39:12,000 --> 00:39:18,000
good enough.  For certain
therapeutic applications,

429
00:39:18,000 --> 00:39:23,000
for example, taking human genes and
cloning them to make therapeutic

430
00:39:23,000 --> 00:39:29,000
proteins or to do gene therapy you
cannot do standard cloning.

431
00:39:29,000 --> 00:39:33,000
And the reason is that human genes
are usually way too big.

432
00:39:33,000 --> 00:39:37,000
They have exons and introns,
which you learned about, and they

433
00:39:37,000 --> 00:39:41,000
can be hundreds of thousands and
sometimes millions of nucleotides in

434
00:39:41,000 --> 00:39:45,000
length.  And that's way too big to
fit into one of these plasmid

435
00:39:45,000 --> 00:39:49,000
cloning vectors and to efficiently
introduce it to bacteria.

436
00:39:49,000 --> 00:39:53,000
And so for the purposes of
generating clones from human genes

437
00:39:53,000 --> 00:39:58,000
we often use a technique
called cDNA cloning.

438
00:39:58,000 --> 00:40:06,000
And I'll take you through this

439
00:40:06,000 --> 00:40:13,000
briefly.  Recall that human DNA,
human genes are broken up into

440
00:40:13,000 --> 00:40:19,000
coding elements that we call exons.
And we usually number these exons

441
00:40:19,000 --> 00:40:26,000
from left to right,
one, two, three.  During the process

442
00:40:26,000 --> 00:40:33,000
of transcription a copy of this gene
is made in the form of a precursor

443
00:40:33,000 --> 00:40:40,000
mRNA which has the same sequence.
It has both the exons and the

444
00:40:40,000 --> 00:40:48,000
introns present.
And then before this is translated

445
00:40:48,000 --> 00:40:56,000
into protein the information in the
introns has to be removed in a

446
00:40:56,000 --> 00:41:02,000
process called splicing.
And that produces a mRNA which

447
00:41:02,000 --> 00:41:08,000
carries the exons lined up next to
each other.  So the introns are

448
00:41:08,000 --> 00:41:14,000
removed, the exons are joined,
and it's this mRNA that then gets

449
00:41:14,000 --> 00:41:20,000
translated into protein.
As I said, this can be very,

450
00:41:20,000 --> 00:41:26,000
very large.  But this is rather
short.  And so if we want to make a

451
00:41:26,000 --> 00:41:32,000
copy of a gene we can also make a
copy of its mRNA.

452
00:41:32,000 --> 00:41:38,000
And that might be more efficient and
more useful.  And this is a

453
00:41:38,000 --> 00:41:44,000
technique called cDNA cloning.
It was made possible by an

454
00:41:44,000 --> 00:41:50,000
invention at MIT,
a discovery at MIT of an enzyme that

455
00:41:50,000 --> 00:41:56,000
was not known to exist,
an enzyme which violated the

456
00:41:56,000 --> 00:42:02,000
so-called central dogma.
The central dogma being that DNA is

457
00:42:02,000 --> 00:42:06,000
transcribed into RNA and that's
translated into protein.

458
00:42:06,000 --> 00:42:11,000
David Baltimore discovered an
enzyme called reverse

459
00:42:11,000 --> 00:42:19,000
transcriptase.

460
00:42:19,000 --> 00:42:24,000
Which can take the information in
RNA and convert it to a DNA form.

461
00:42:24,000 --> 00:42:29,000
And he actually won the Nobel Prize
for that.  So this is the principle.

462
00:42:29,000 --> 00:42:34,000
You can take an mRNA which has
polarity 5 prime to 3 prime.

463
00:42:34,000 --> 00:42:39,000
This mRNA that I produced up there,
for example.

464
00:42:39,000 --> 00:42:44,000
You can anneal onto it an
oligonucleotide primer.

465
00:42:44,000 --> 00:42:49,000
This oligonucleotide primer is made
of DNA.  It has a 5 prime end and a

466
00:42:49,000 --> 00:42:54,000
3 prime end.  And then you add this
enzyme reverse transcriptase,

467
00:42:54,000 --> 00:43:00,000
which I'll abbreviate RT.
Reverse transcriptase is unique in

468
00:43:00,000 --> 00:43:06,000
its ability to copy from an RNA
template a DNA strand.

469
00:43:06,000 --> 00:43:12,000
And so you'll get a duplex which
has RNA on the top and

470
00:43:12,000 --> 00:43:20,000
DNA on the bottom.

471
00:43:20,000 --> 00:43:29,000
You then get rid of the RNA.

472
00:43:29,000 --> 00:43:40,000
You hydrolyze the RNA.

473
00:43:40,000 --> 00:43:45,000
So all you're left with is that
single strand of DNA.

474
00:43:45,000 --> 00:43:50,000
You now anneal on another primer,
another oligonucleotide primer which

475
00:43:50,000 --> 00:43:55,000
now has a polarity 3 prime to 5
prime in this direction because this

476
00:43:55,000 --> 00:44:00,000
one went 5 prime to 3 prime in this
direction.  And then you

477
00:44:00,000 --> 00:44:07,000
add DNA polymerase.

478
00:44:07,000 --> 00:44:12,000
Which will extend from this primer
and make a full duplex of DNA where

479
00:44:12,000 --> 00:44:17,000
both strands are made out of DNA.
And this we call a cDNA clone, a

480
00:44:17,000 --> 00:44:22,000
copy of the mRNA.
cDNA clone.  And you can make

481
00:44:22,000 --> 00:44:27,000
libraries of cDNA clones from all
the RNAs in your cells,

482
00:44:27,000 --> 00:44:32,000
for example.
And then you can look,

483
00:44:32,000 --> 00:44:36,000
using various techniques,
at different clones within that

484
00:44:36,000 --> 00:44:40,000
library for ones that function in
different ways.

485
00:44:40,000 --> 00:44:44,000
So I want to take you through a
couple of examples of how we use

486
00:44:44,000 --> 00:44:49,000
cDNAs in the context of gene therapy.
This one comes out of your book.

487
00:44:49,000 --> 00:44:53,000
This is an application where
there's an enzyme called TPA which

488
00:44:53,000 --> 00:44:57,000
will dissolve clots.
It's called tissue plasminogen

489
00:44:57,000 --> 00:45:02,000
activator.
And it's used now to treat people

490
00:45:02,000 --> 00:45:06,000
who've had heart attacks or strokes.
So what was done was to make a cDNA

491
00:45:06,000 --> 00:45:10,000
copy of the TPA gene,
introduce that into a plasmid vector

492
00:45:10,000 --> 00:45:14,000
with appropriate sequences in red
and yellow to allow the expression

493
00:45:14,000 --> 00:45:18,000
of that gene in bacteria,
transfer it into E. coli by

494
00:45:18,000 --> 00:45:22,000
transformation.
Now, the E. coli will pump out this

495
00:45:22,000 --> 00:45:27,000
enzyme TPA.
You can then purify that enzyme and

496
00:45:27,000 --> 00:45:32,000
inject it into this happy looking
stroke patient to help dissolve the

497
00:45:32,000 --> 00:45:37,000
clots that it formed in the
formation of the stroke.

498
00:45:37,000 --> 00:45:41,000
OK?  So that's an example of
genetic engineering one of our genes

499
00:45:41,000 --> 00:45:46,000
into bacteria to turn them into
little protein factories from which

500
00:45:46,000 --> 00:45:51,000
you can purify the enzyme and
hopefully mitigate the consequences

501
00:45:51,000 --> 00:45:59,000
of the disease.

502
00:45:59,000 --> 00:46:03,000
A second example,
as opposed to putting in a

503
00:46:03,000 --> 00:46:07,000
therapeutic protein,
you might want to put in a

504
00:46:07,000 --> 00:46:11,000
therapeutic gene.
So the example here I'll give you

505
00:46:11,000 --> 00:46:17,000
is cystic fibrosis.

506
00:46:17,000 --> 00:46:21,000
I've mentioned to you in earlier
lectures that cystic fibrosis is a

507
00:46:21,000 --> 00:46:26,000
genetic disease in which the
individuals have a defect in a

508
00:46:26,000 --> 00:46:31,000
transporter, an ion transporter,
so that in contrast to normal cells,

509
00:46:31,000 --> 00:46:35,000
here is a normal cell which has this
CFTR transporter which will allow

510
00:46:35,000 --> 00:46:40,000
chloride ions to move in and out of
the cell, as is a wild

511
00:46:40,000 --> 00:46:45,000
type individual.
In the case of CF,

512
00:46:45,000 --> 00:46:50,000
that transporter is missing.
That leads to an inability of the

513
00:46:50,000 --> 00:46:54,000
cells to properly regulate their
water content,

514
00:46:54,000 --> 00:46:59,000
and this leads to disease.
So what could you do about this

515
00:46:59,000 --> 00:47:04,000
disease?
Well, why don't you just put the

516
00:47:04,000 --> 00:47:09,000
defective gene back in?
We could make a cDNA copy of the

517
00:47:09,000 --> 00:47:14,000
cystic fibrosis gene,
build it into a vector and introduce

518
00:47:14,000 --> 00:47:19,000
it not into bacteria but back into
these cells.  So we could clone a

519
00:47:19,000 --> 00:47:24,000
CFTR cDNA back into these cells.
They would, in fact, pick up that

520
00:47:24,000 --> 00:47:30,000
DNA and begin to express it.
They would make this protein.

521
00:47:30,000 --> 00:47:34,000
And that would allow them to
transport chloride properly.

522
00:47:34,000 --> 00:47:39,000
And they might be ìnormalî.  This
actually works extremely well in the

523
00:47:39,000 --> 00:47:43,000
tissue culture dish.
It doesn't work nearly so well in

524
00:47:43,000 --> 00:47:48,000
the context of a human being.
Even though we can make viral

525
00:47:48,000 --> 00:47:53,000
versions of this,
viruses that carry cDNAs,

526
00:47:53,000 --> 00:47:58,000
recombinant viruses, and I'll draw
that up here.

527
00:47:58,000 --> 00:48:07,000
Using the same methods of

528
00:48:07,000 --> 00:48:12,000
recombinant DNA technology,
I can make a recombinant virus,

529
00:48:12,000 --> 00:48:17,000
like a cold virus that now carries a
piece of this cDNA for CFTR,

530
00:48:17,000 --> 00:48:23,000
and I could use that to infect a
person who has CF.

531
00:48:23,000 --> 00:48:31,000
That's a person and they have CF so

532
00:48:31,000 --> 00:48:37,000
they're not happy.
I could introduce into their nasal

533
00:48:37,000 --> 00:48:43,000
passage, they could breath in this
virus that carries the gene.

534
00:48:43,000 --> 00:48:48,000
And if the gene, if the virus
infected the cells of the lung,

535
00:48:48,000 --> 00:48:54,000
remember the problem occurs in the
lung, maybe, if this were very

536
00:48:54,000 --> 00:49:00,000
efficient, the person would be happy,
we could cure them.

537
00:49:00,000 --> 00:49:04,000
The problem is that this
introduction of the virus is very

538
00:49:04,000 --> 00:49:08,000
inefficient in vivo,
in the person.  It doesn't work very

539
00:49:08,000 --> 00:49:12,000
well.  You cannot get enough virus
in to infect enough cells to correct

540
00:49:12,000 --> 00:49:17,000
the disease in the person.
So although gene therapy is very

541
00:49:17,000 --> 00:49:21,000
attractive for many diseases such as
CF, it actually hasn't worked very

542
00:49:21,000 --> 00:49:25,000
well.  But there is one example of a
cure using gene therapy.

543
00:49:25,000 --> 00:49:30,000
And I want to just end by telling
you that story.

544
00:49:30,000 --> 00:49:33,000
To cure the disease you probably
heard about, the disease that causes

545
00:49:33,000 --> 00:49:36,000
ìthe boy in the bubbleî syndrome,
which is severe combined

546
00:49:36,000 --> 00:49:39,000
immunodeficiency,
or at least one form of it,

547
00:49:39,000 --> 00:49:42,000
these individuals have a defect in
an enzyme called ADA,

548
00:49:42,000 --> 00:49:46,000
adenosine deaminase.  And this
results in a very severe

549
00:49:46,000 --> 00:49:49,000
immunodeficiency.
They don't have their immune cells.

550
00:49:49,000 --> 00:49:52,000
They cannot fight infection.  So
the kids have to stay inside

551
00:49:52,000 --> 00:49:55,000
protective chambers to avoid
exposure to pathogens,

552
00:49:55,000 --> 00:49:59,000
the bubble.  For a long time they
really weren't well treated.

553
00:49:59,000 --> 00:50:02,000
They could be treated with having a
little bit of the enzyme.

554
00:50:02,000 --> 00:50:05,000
I guess that's not shown here but
on the next slide.

555
00:50:05,000 --> 00:50:09,000
If you add the enzyme back,
even to their blood, you can get

556
00:50:09,000 --> 00:50:12,000
some protection.
But, nevertheless,

557
00:50:12,000 --> 00:50:16,000
the kids did not do terribly well
and often died from infections.

558
00:50:16,000 --> 00:50:19,000
Here's the disease mechanism.  It's
caused by a defective enzyme that

559
00:50:19,000 --> 00:50:23,000
normally breaks down adenosine.
This leads to a buildup of a

560
00:50:23,000 --> 00:50:26,000
precursor, or rather a metabolite
that kill cells of the immune system,

561
00:50:26,000 --> 00:50:30,000
and the individuals have immunocompromised
state.

562
00:50:30,000 --> 00:50:34,000
If you can just hang on for one or
two more minutes.

563
00:50:34,000 --> 00:50:38,000
So what's done here,
in contrast to the failed example

564
00:50:38,000 --> 00:50:42,000
for CF where I said that it's very
difficult to get the virus into the

565
00:50:42,000 --> 00:50:46,000
cells in the person,
what's done in the case of this

566
00:50:46,000 --> 00:50:51,000
disease is to take the cells out of
the person.  And this is called ex

567
00:50:51,000 --> 00:50:55,000
vivo gene therapy where you isolate
cells from the bone marrow of one of

568
00:50:55,000 --> 00:50:59,000
these kids, the stem cells that give
rise to the cells of the blood

569
00:50:59,000 --> 00:51:03,000
system, and then you infect those
cells in vitro with the virus that

570
00:51:03,000 --> 00:51:07,000
carries the cDNA of the ADA gene.
You isolate those cells that have

571
00:51:07,000 --> 00:51:11,000
that gene and then you introduce the
cells back into the person.

572
00:51:11,000 --> 00:51:15,000
And you can control that process
much more carefully,

573
00:51:15,000 --> 00:51:18,000
much more efficiently,
and you have a much greater

574
00:51:18,000 --> 00:51:22,000
concentration of cells that are
doing the right thing now.

575
00:51:22,000 --> 00:51:26,000
This goes through it in some more
detail.  It's a version of a slide

576
00:51:26,000 --> 00:51:30,000
from your book so you
can look at it.

577
00:51:30,000 --> 00:51:34,000
It's gene therapy.
And I want to point out that

578
00:51:34,000 --> 00:51:39,000
although it can work,
and it does work, it has one risk,

579
00:51:39,000 --> 00:51:43,000
namely that the virus that carries
the therapeutic gene actually

580
00:51:43,000 --> 00:51:48,000
integrates into the DNA of the cells.
And if it integrates into a gene

581
00:51:48,000 --> 00:51:52,000
that's important that integration
could actually cause a mutation.

582
00:51:52,000 --> 00:51:57,000
So there's a risk associated with
gene therapy.

583
00:51:57,000 --> 00:52:00,000
And, in fact, although this was
successful, as you can see here,

584
00:52:00,000 --> 00:52:04,000
two kids with SCID, or actually a
number of kids with SCID were cured

585
00:52:04,000 --> 00:52:08,000
through this ex vivo gene therapy
approach, and now they can run

586
00:52:08,000 --> 00:52:12,000
around like everyday kids,
unfortunately, in this first study

587
00:52:12,000 --> 00:52:16,000
of 11 patients,
all of whom were ìcuredî,

588
00:52:16,000 --> 00:52:20,000
later two of the kids ended up
getting leukemia.

589
00:52:20,000 --> 00:52:24,000
And they did because the virus had
inserted itself,

590
00:52:24,000 --> 00:52:28,000
the genome of the virus had inserted
itself next to a gene that when it

591
00:52:28,000 --> 00:52:32,000
becomes too active it causes the
cells of the immune system to

592
00:52:32,000 --> 00:52:36,000
proliferate abnormally
leading to leukemia.

593
00:52:36,000 --> 00:52:40,000
So although there was a benefit
there was also a risk.

594
00:52:40,000 --> 00:52:45,000
In this case, families of these
kids actually are willing to take

595
00:52:45,000 --> 00:52:49,000
that risk because it's such an awful
disease.  But you have to be aware

596
00:52:49,000 --> 00:52:54,000
that in all of these kinds of
therapies there's a risk-benefit

597
00:52:54,000 --> 00:52:57,000
analysis that has to go on.