>> GOOD AFTERNOON.
I'M PAUL SEVING, DIRECTOR OF THE
NATIONAL EYE INSTITUTE, IT'S MY
PLEASURE TO WELCOME FRED GAGE TO
THIS AFTERNOON LECTURE AT NIH.
ACTUALLY FRED IS BETTER KNOWN AS
RUSTY.
RUSTY IS HEAD OF LABORATORY OF
GENETICS AT THE SALK INSTITUTE.
HE RECEIVED A Ph.D. FROM JOHNS
HOPKINS IN 1976 AND THEN THEN
TRAVELED TO TEXAS, SWEDEN.
AND THIN THE GNASH
AGED NEURODEGENERATIVE DISEASES.
RUSTY'S WORK CENTERS AROUND THE
CENTRAL NERVOUS SYSTEM AND THE
UNEXPECTED PLASTICITY THAT THE
CNS EXHIBITS THROUGHOUT THE LIFE
OF ALL MAMMALS.
HIS STUDY USES CELLULAR TECH
NIEKS ON ENVIRONMENTAL
INFLUENCES THAT HELP REGULATE
NEUROGENESIS IN THE ADULT.
OF COURSE THE NATIONAL EYE
INSTITUTE IS PLEASE THAT YOU
HAVE FOUND YOUR WAY TO THE
NEURAL RETNA AND HAVE SEEN
THAT'S A GOOD SYSTEM TO EXPLORE
THESE IDEA.
HE'S WON NUMEROUS PRIDES IN HIS
WORK, A VERY BRIEF LIST IS THE
KO SCIENCE PRIZE, THE CHARLES
DANA AWARD, METROPOLITAN AWARD
AND THE EPISON PRIZE FOR
NEUROPLASTICITY.
HE'S A TRIPLE THREAT IN THAT HE
HOLDS MEMBERSHIP, WAS ELECTED TO
MEMBERSHIP IN THE NATIONAL
ACADEMY OF SCIENCES, THE
INSTITUTE OF MEDICINE AND
NATIONAL ACADEMY OF SCIENCES.
ANOTHER IS HE'S PREVIOUS LISTEN
BY PRESIDENT FOR SOCIETY FOR
NEUROSCIENCE AND PRESIDENT ELECT
OF THE INTERNATIONAL SOCIETY FOR
STEM CELL RESEARCH.
PERHAPS FOR US TODAY A FINAL
MEASURE OF HIS WORK ON
PLASTICITY IS THAT HE'S
DEMONSTRATING THAT TODAY AND
GIVEN THE WAL IS LECTURE THE
WEDNESDAY AFTERNOON LECTURE ON
THURSDAY.
SO PLEASE WELCOME DR. GAGE IN
NEURAL PLASTICITY IN THE ADULT
MAMMALIAN BRAIN.
[APPLAUSE]
>> THANK YOU VERY MUCH FOR THAT
KIND INTRODUCTION.
PLEASURE TO BE BACK.
I HAVEN'T BEEN BACK TO NIH IN A
WHILE.
AND WHILE I WAS THINKING ABOUT
THE TYPE OF LECTURE THAT I WOULD
GIVE HERE I DECIDED THAT I WOULD
GIVE A DUAL TALK, ONE A BRIEF
UPDATE ON THE WORK WE HAVE BEEN
SPENDING MUCH OF OUR TIME THE
LAST 15 YEARS DOING ON ADULT
NEUROGENESIS AND PLASTICITY, TO
REFRESH Y'ALL WHAT I'M
INTERESTED IN.
IN THAT REALM WHERE WE'RE GOING.
BUT THEN I'LL DIVERGE A BIT AND
TALK ABOUT SOMETHING THAT WE'RE
SPEND MORGUE TIME ON IN MY
LABORATORY SPINNING OUT OF WORK
THAT INITIALLY WAS STARTED AS A
FUNCTION OF OUR WORK IN ADULT
NEUROGENESIS.
I THINK STUDENTS AND POST-DOCS
IN THE LAB MAYBE JUNIOR
INVESTIGATORS, MAYBE EVEN SENIOR
INVESTIGATORS, IT'S AN
INTERESTING LESSON PRORKING ON
ONE PROJECT, GOING DEEP TO TRY
TO UNDERSTAND MORE ABOUT IT AND
THEN YOU HAVE SOME SERENDIPITOUS
FINDENING YOUR LAB AND IT LOOKS
INTERESTING AND YOU THINK TO
YOURSELF, SHOULD I FOLLOW THIS
UP?
IT'S KIND OF RISKY, OR SHOULD I
STICK WITH WHAT I'M DOING.
WHERE IT'S SAFE AND I CAN GET
SOME SUCCESS.
SO WE STARTED THIS SECOND PART
OF MY TALK ABOUT EIGHT YEARS
AGO.
AND I'LL TELL YOU ABOUT THIS
TRANSITION POINT AND WHERE WE
ARE ON THIS JOURNEY OF TRYING TO
FIND OUT SOMETHING SOMETHING WE
OR I DIDN'T KNOW MUCH ABOUT AT
ALL.
THE FIRST PART OF THIS TALK
DEALS WITH -- I MUST SAY THAT
WHERE I AM IN THIS OTHER AREA IS
REMINISCENT HOW I STARTED IN
THIS AREA OF ADULT NEUROGENESIS
WHERE EARLY ON THE QUESTION WAS
WHETHER OR NOT THERE REALLY ARE
CELLS DIVIDING IN THE ADULT
NERVOUS SYSTEM AS THIS HAD NOT
BEEN ACCEPTED WELL AS A
UNDERLYING PRINCIPLE WITHIN
ADULT NEUROBIOLOGY.
REASON FOR THIS LACK OF
ACCEPTANCE IS BECAUSE MANY NOT
BEING NEUROBIOLOGISTS ACCEPT THE
REALIZATION THAT THE BRAIN IS
COMPLICATED, MORE COMPLICATED
THAN ANY OTHER ORGAN BUT AT
LEAST YOU KNOW THAT THE
PRINCIPLE CELLS IN THE CENTRAL
NERVOUS SYSTEM ARE THESE
NEURONAL STRUCTURES WITH LARGE
CELLS WITH MANY PROCESSES AND
OUTRAGEOUS NUMBERS WE GIVE TO
THEM, HUMANS HAVING ON THE ORDER
OF 100 BILLION NEURONS AND
100 TRILLION CONNECTIONS BETWEEN
THEM.
AND THE QUESTION WAS HOW COULD
-- AND ALSO THE BRAIN BEING
STRUCTURED WHICH IS INVOLVED IN
OUR SENSORY AWARENESS, OUR
ABILITY TO THINK AND COMMUNICATE
WITH EACH OTHER, SO THE IDEA
THAT THERE WERE CELL OR NEURONS
IN THE BRAIN THAT COULD DIVIDE
AND GIVE RISE TO NEW NEURONS WAS
MET MET WITH CONCERNS AND
SKEPTICISM THAT A CELL THIS
COMPLEX COULD DIVIDE AND GIVE
RISE TO ANOTHER CELL WITH
CONNECTIONS.
THE TRUTH IS THAT THAT DOESN'T
HAPPEN IN THE ADULT NERVOUS
SYSTEM BUT RATHER THERE ARE
STILL CELLS THAT PERSIST IN THE
ADULT NERVOUS SYSTEM THROUGHOUT
LIFE.
SO THAT'S ONE OF THE ISSUES SO
NEURONS DON'T DIVIDE.
OTHER AREAS THEY DON'T DIVIDE
EVERYWHERE.
SO CELLS ARE NOT JUST DIVIDING
EVERY IN THE ADULT BRAIN BUT THE
RESTRICTED TO TWO AREAS OF THE
BRAIN.
ONE AREA, STRUCTURE CAWBED SUB
VENTRICULAR ZONE, THE LINING OF
SUB VENTRICULAR ZONE IS MADE UP
OF A NICHE OF CILIATED CELL AND
PROGENITOR AND GLIAL CELLS THAT
PERSIST THROUGHOUT LIFE IN THIS
SUB VENTRICULAR STRUCTURE,
PERIODICALLY THE NEURAL
PROGENITOR CELL MIGRATES OVER A
LOOK DISTANCE IN FACT TO THE
OLFACTORY BULB WHERE THEY TURN
PERPENDICULARLY AND
DIFFERENTIATE INTO GRANULOCELLS,
SOME DO, AND THE REST BYPASS
THROUGH THIS -- THESE LARGE
MITRAL ENTUFTED CELLS TO THE
GLAMIUOS WHERE THEY
DIFFERENTIATE TO NEURONS.
THIS PROCESS CONTINUES
THROUGHOUT LIFE AND IN MICE THEY
HAVE DONE SOME COLLEAGUES IN
JAPAN HAVE DONE AN INTERESTING
STUDY DOING LINEAGE TRACING SAW
SAYING THE STRUCTURE TURNS OVER
1 MILLION TIMES IN THE ADULT
MOUSE.
A SIGNIFICANT TURN OVER.
THE OTHER AREA THIS OCCURS AND
WHERE I SPENT MOST EFFORT IS IN
THE STRUCTURE CALLED THE
HIPPOCAMPUS AND THIS IS AN AREA
OF THE BRAIN INVOLVED IN
LEARNING AND MEMORY.
PARTICULARLY IN THE ACQUISITION
OF NEW MEM RIS AN RECOLLECTION
OF OBJECTS RELATED TO EACHER.
SO THE FUNCTION IS INVOLVED IN
PATTERN SEPARATION.
HIPPOCAMPUS TAKES INFORMATION
FROM LOTS OF SENSORY MODAL
DITIES AND BRINGS THEM TO THE
HIPPOCAMPUS WHERE IT'S STORED IN
THE CORTEX.
THE THESE NEWLY BORN CELLS OCCUR
IN THE DENT ATE WHERE THEY SIT
AT THE BASAL AREA AND THEY
PROLIFERATE IN CLUSTERS AND GIVE
RISE TO NEWBORN NEURONS SOME
WHICH DIE, OTHERS INTEGRATE TO
CIRCUITRY.
WE NOW KNOW IN ALL STRUCTURES
AND MAMMALS TO DATE HAVE SHOWN
NEUROGENESIS AT LEAST IN THE
HIPPOCAMPUS.
THERE STILL REMAINS SOME
QUESTION IN THE SUB VENTRICULAR
ZONE OF THE OLFACTORY BULB
WHETHER OR NOT NEUROGENESIS
OCCURS BUT IN THE HIPPOCAMPUS IT
DOES OCCUR.
THIS IS A VARIETY OF WAYS ONE
MONITORS THIS.
EARLY ON WE USED BROAM DEOXI
URIDINE USED IN CANCER STUDIES
TO LABEL PROLIFERATING CANCERS
BUT THAT IT DOES GO TO CENTRAL
NERVOUS SYSTEM WELL AND
INCORPORATED INTO THE DNA
DIVIDING CELLS.
THIS ALLOWS AS A FATE MARKER TO
SEE WHICH CELLS SURVIVED AND
WHERE THEY R. HERE IS AN ADULT
RAT TWO MONTHS AFTER INJECTION
OF BRDU IN EACH OF THESE BLACK
DOTS, SHOW AS NEWLY BORN CELL
BORN IN THE ADULT ANIMAL AND
SURVIVED AT LEAST TWO MONTHS.
THERE IS A WAY TO QUANTITATE HOW
MANY NEW CELLS ARE COMING TO THE
SYSTEM BUT WE HAVE MORE RECENTLY
DEVELOPED THE USE OF A
RETROVIRUS AS A WAY TO TAG THE
THE CELLS GENETICALLY SINCE THE
MILLENNIUM INTEGRATES INTO CELLS
THAT ARE UNDERGOING CELL
DIVISION BECAUSE IT DOESN'T HAVE
AN IMPORT MACHINERY TO IT, YOU
CAN USE THIS TO CATCH DIVIDING
CELLS AND THEN LABEL THEM.
THIS WAY YOU CAN OBSERVE THE
DENDRITES IN THE SPINES AND USE
THIS VECTOR AS WELL TO KNOCK
DOWN GENES OR OVEREXPRESS GENES
SO IT'S A USEFUL TOOL FOR
FOLLOWING ACTIVITY, THREE DAYS
AFTER INJECTION OF THE FLAT
POLARIZED IN A HORIZONTAL
MANNER, BY SEVEN DAYS THEY
ORIENT.
BY 14 DAYS THEY HAVE POLARIZED
INTO THIS AREA CALLED THE
MOLECULAR AIR AND THEY SEN THEIR
AXON OUT TO THE TARGET AREA
WHICH IS THE CA-3 AND BY 21 DAYS
THEY HAVE SPINES AND ARE
STARTING TO MAKE MORPHOLOGICAL
CONNECTS WITH EACH OTHER.
BY DAY 28 THEY ARE
ELECTROPHYSIOLOGICALLY ACTIVE,
YOU CAN PATCH CLAMP THE CELLS
AND SHOW THEY'RE ACTIVE.
THE PROCESS OF NEUROGENESIS IS
NOT A NEURON IS BORN BUT RATHER
GOES THROUGH QUITE A RANGE OF
CHANGES AND DOESN'T FULLY MATURE
FOR ANOTHER MONTH SO IT TAKES
EIGHT WEEKS FOR THESE CELLS, THE
CURRENT THINKING OF THIS IN THE
PERIOD BETWEEN 21 DAYS AND 48
DAYS WHEN CELLS ARE UNIQUELY
ACTIVE AND THEY'RE
HYPEREXCITABLE AT THIS STAGE.
THAT'S AN IMPORTANT FEATURE IN
WHY THEY PERSISTED THROUGHOUT
LIFE IN ALL THESE MAMMALIAN
CELLS.
THIS IS A PICTURE OF -- WE THINK
ABOUT WHY -- HOW COULD THIS
SMALL NUMBER OF NEW CELLS BEING
GENERATE IN A STRUCTURE IMPACT
ON BEHAVIOR.
EEF ONE OF THESE GREEN CELLS
CORRESPONDS TO A CELL BORN IN
THE DENTTE OF ADULT ANIMAL
WITHIN A THREE AND A HALF HOUR
PERIOD OF TIME BECAUSE THE
SINGLE INJECTION OF A VIRUS
THAT'S FIVE TIMES TEN TO THE
SEVENth AND IT DEGRADES
RAPIDLY SO THESE CELL WERE BORN
TWO MONTHS AGO, THIS IS A
SECTION THROUGH THE ENTIRE
HIPPOCAMPUS REPRESENTING NEW
CELLS.
HERE THEY HAVE SURVIVED AT LEAST
TWO MONTHS.
THEY HAVE THE NICE DENDRITES
RECEIVED INPUT FROM AROUND THE
CORTEX AND ALL OF THIS
CORRESPONDS TO THE AXONS HERE
YOU GET A BETTER IMAGE HOW THEY
IMPACT THE WHOLE STRUCTURE
ITSELF.
THIS PROCESS OF NEUROGENESIS IS
NOTS STABLE THROUGHOUT LIFE BUT
RATHER IS HIGHLY REGULATED BY A
VAIRT OF EVENTS INCLUDING
PHYSICAL ACTIVITY VARIETY OF
NEUROGENESIS CAN INCREASE, AND
EVERY MODEL I LOOKED AT THERE'S
DECREASE IN NEUROGENESIS, IN
HUMAN ANIMALS BUT THE PROBLEM IS
WE DON'T KNOW YET WHETHER OR NOT
THIS DECREASE IN NEUROGENESIS IN
SOME OF THESE ANIMAL MODELS FOR
EXAMPLE IS CAUSED BY THE
MUTATIONS OR WHETHER OR NOT IT'S
CAUSED BY SOME CHANGE IN THE
BEHAVIOR OF THE ANIMAL THAT
CONSEQUENTLY RESULTS IN A
DECREASE.
ONE OF THE MOST ROBUST PHENOM
NONA ONE HAS SEEN IN THIS AREA
WAS SOMETHING HENRY VAN PRAGUE
DISCOVERED IN OUR LAB MANY YEARS
AGO.
IF YOU TAKE A NORMAL HEALTHY
MOUSE AND PUT THEM ON A
TREADMILL ACTUALLY PUT THEM IN
RUNNING WHEELS, THIS IS
MANUFACTURED, AND WE'VE USED OUR
MAC TO PHOTO SHOP THIS.
THIS IS NOT A REAL TILE, WE
DON'T ACTUALLY MAKE THAT BUT
THERE'S A LINEAR CORRELATION
BETWEEN THE NUMBER OF -- THE
DISTANCE RUN AND THE NUMBER OF
NEW CELLS, THIS HOLDS UP, TURNS
OUT IN A VARIETY OF SPECIES.
AND ALSO MORE RECENTLY A LOT OF
WORK BEING DONE IN HUMANS
SHOWING CHANGES IN HIPPOCAMPAL
VOLUME WITH PHYSICAL EXERCISE.
SO THERE'S A WHOLE AREA OF
RESEARCH THAT CONTINUES ON THIS
VEIN BUT NOW I'M GOING TO START
TAKEING YOU OFF IN THIS OTHER
TANGENT.
AND IT BEGAN AS WE DEVELOPED
PROCEDURES TO ISOLATE THESE
CELLS FROM THE ADULT NERVOUS
SYSTEM, TO STUDY THEIR
PROPERTIES IN VITRO.
OUR OBJECTIVE HERE WAS TO LEARN
MORE ABOUT THE CELL AND
MOLECULAR BIOLOGY HOW THESE
CELLS BEHAVE.
WE DEVELOPED TECHNIQUES TO
ISOLATE CELLS WITH GRADIANT FROM
ANYWHERE IN THE ADULT BRAIN.
PULL DOWN A POPULATION, FEED
THEM INTO CULTURE, WITH FGF-2
UNDER A SERUM FREE CONDITION.
WHEN WE DO THAT THEY GROW
INDEFINITELY BASICALLY AS STEM
CELLS AND THIS IS HOW WE
DETERMINE THEY WEREN'T STEM
CELLS BY VIRTUE OF ABILITY TO
SELF RENEW AND GIVE RISE TO
NEURAL IMAGES BUT THESE CELLS
WERE ALSO CLONABLE SO WE CAN
TAKE INDIVIDUAL CLONES AND PUT
THEM IN A DISH B AND THEY'LL
GROW AS CLONES.
WE USE THE RETROVIRUS TITER TO
PULL OUT CLONES THAT ARE
DIFFERENT COLOR AND THEN WE CAN
DO SINGLE CELL, EXPAND THE CLONE
UP INAND DO A SOUTHERN BLOT AND
SHOW THAT THE EXPANDED CLONE IS
ALL DERIVED FROM THE CELL WITH A
SINGLE INTEGRATION SITE.
SO THIS IS OUR WAY OF CLONALLY
MOLECULAR CLONING OF THE CELLS.
WE CAN DO THIS INDEFINITELY WITH
MULTIPLE CLONES CONFIRMING THAT
THEY DO HAVE P STEM CELL
PROPERTIES.
WE ALSO ESTABLISH CONDITIONS
WHERE WE CAN TAKE INDIVIDUAL
CELLS AND CULTURE AND THEY'LL
DIVIDE OR THEY'LL SURVIVE AND IN
THIS CASE IT'S WITH WITH FGF-2
AND A PROTEIN THAT WE HAD
ISOLATEED FROM THE CONDITION
MEDIA THAT WHEN WE GIVE IT BACK
TO THE CELLS IT MAINTAINS THEM
AT THIS LOW DENSITY AND THEY'LL
PERSIST AS THESE CELLS FOR LONG
PERIODS OF TIME AND THEY'LL
DIVIDE AND YOU CAN SAY THEY'RE
DIVIDING SYMMETRICALLY AND
RETAINING THEIR STEM CELL
PROPERTIES SO THEY ARE
SELF-RENEWING.
DOWN HERE, I DON'T KNOW IF YOU
CAN SEE IT THE HOURS, THIS IS
THE NUMBER OF HOURS IT TAKESCH
THESE CELLS DIVIDE INDEFINITELY.
UNTIL THEY REACH A POINT WHERE
THEY'RE CONFLUENT, THEY COME IN
CONTACT WITH EACH OTHER AND
DIFFERENTIATE.
WHAT WE WANTED TO KNOW WHAT WERE
THE MOLECULES THAT WERE
IMPORTANT, THE -- DEFINE THESE
CELLS IN THESE DIFFERENT STATES.
WE DEVELOPED CONDITIONS WHERE WE
COULD TAKE THE STEM CELL WE
COULD DEFINE BY A VARIETY OF
FACTORS AN MAIN THE STEM CELLS
WITH THESE FACTORS AN SWAP TO
OTHER CONDITIONS WE CAN MAKE A
PURIFIED POPULATION OF NEURONS,
OLIGODENDROCYTES AND ASTROCYTES.
AND WITH THE CCG WE CAN KEEP
THEM IN A VERY SPECIAL
CONDITION.
SO BY VIRTUE OF BEING ABLE TO
DIFFERENTIATE THE CELLS INTO ALL
THESE DIFFERENT LINEAGES AS WELL
AS MAINTAIN THEM AS STEM CELLS,
WE DEVELOPED A -- WE DEVELOPED
-- SET UP EARLY MICROARRAY, IN
THESE EARLY DAYS USE GLASS
MICROARRAYS AS SOME OF YOU MAY
REMEMBER AND YOU HAD TO MAKE
YOUR OWN.
IN THOSE DAYS THE ARRAYS WERE
MADE UP OF COMPLETE GENOMIC
MATERIAL SO WE DIDN'T MASK OR
REPEAT AREAS.
THIS IS PRETTY MUCH EVERYTHING
THAT WE WERE SCANNING FOR, THAT
MIGHT BE EXPRESSED WITHIN THESE
CELLS AND THEIR DIFFERENT
LINEAGES.
A SPECIAL LINEAGE WE WERE
LOOKING AFTER IS ONE THAT WAS
FGF-2, CCG, THESE CELLS WERE
COMMITTED TO BECOME NEURONS BUT
THEY WERE STILL DIVIDING SO THIS
PURIFIED LOW DENSITY NEUROGENIC
POPULATION WE WERE PARTICULARLY
INTERESTED IN, WE DID THE
ARRAYS, FOR THIS POPULATION AND
SUBTRACTED THE GENES EXPRESSED
VERSUS NEURONS, ASTROCYTES,
OLIGODENDROCYTES AS WELL AS
AUTHENTIC HETEROGENEOUS STEM
CELL POPULATION.
AND THE GENES THAT CAME UP IN
THE FIRST NINE GENES E PRESSED
TOP ARRAYS IN THE ARRAY, WERE
THESE SEQUENCES HERE.
SO PARTS OF LINE ELEMENTS OR TWO
WITH THE ENDONUCLEASE COMPONENT.
AND VIN WAS DISAPPOINTED IN THIS
BECAUSE SHE WAS LOOKING FOR
CONONCAL WINT SIGNALS OR NOTCH
SIGNALING OR BNPs THAT MIGHT
BE OF SIGNIFICANCE AND WHAT SHE
GOT WAS WHAT HAD PREVIOUSLY BEEN
CONSIDERED JUNK BY SOME AND BUT
MOBILE ELEMENTS NEVERTHELESS.
WHAT ARE LINE ELEMENTS?
WE HAVE TO GIVE CREDIT AMONG
OTHERS TO THINKING ABOUT THESE
GENOMIC DNA TO BARBARA
MCCLINTOCK WHO IN THE LATE 40s
IDENTIFIED THE FACT THAT THERE
WERE TRANSPOSEONS MOBILE WITHIN
THE DNA OF MAYS.
AND A SIGNIFICANT AMOUNT OF RECK
IN ADDITION WAS GIVEN OVER THE
YEARS BUT THERE WAS ALSO SOME
RESISTANCE TO THIS -- THE
IMPORTANCE THAT THIS MIGHT PLAY.
AND READING BACK THROUGH SOME OF
HER WRITING IT WAS CLEAR THAT IN
ADDITION TO JUST THE
DEMONSTRATION TECHNICALLY OF THE
MOBILITY OF THESE GENES IN THE
GENOME SHE EYE POT SIZED THAT
THESE MOBILE ELEMENTS MAY PLAY
SOME ROLE IN DEVELOPMENT, MAY
PLAY SOME ROLE IN GENE
REGULATION.
IT WAS THIS THAT WAS MET WITH
MORE RESISTANCE THAN THE FACT
THAT THEY WERE JUMPING.
I THINK THIS PART IS SOMETIMES
MISUNDERSTOOD.
LESLIE ORGILL AND FRANCIS CRICK
DESCRIBED A LOT OF HIGH GENOMIC
MATERIAL, JUNK DNA AND RICHARD
DOKINS DISDISCUSSION OF THE
SELFISH GENE CAST AS LIGHT ON
MOBILE ELEMENTS LESS INTERESTING
THAN THEY MIGHT BE TO OTHERS.
BUT WE LOOK AT THE SEQUENCES OF
THE HUMAN GENOME AND LOOK AT THE
TOTAL AMOUNT OF DNA THAT'S
ATTRIBUTABLE TO THESE ELEMENTS.
THE NON-CODING -- THE NON-LTR
LINE ELEMENT ACCOUNT FOR 17% OF
THE ENTIRE DNA WITHIN THE HUMAN
GENOME.
2% IS CODEINE SEQUENCE.
AND MANY OF THESE OTHER
SEQUENCES LIKE ALLUDES, GENES
DNA TRANSPOSEON, ACCOUNT FOR
TOTALLY OVER 50% OF THE TOTAL
GENOME IS MADE UP OF ELEMENTS,
THESE REPEAT ELEMENTS THAT ARE
ONE WAY OR ANOTHER ATTRIBUTABLE
TO THESE MOBILE ELEMENTS WITHIN
THE GENOME.
THE EVOLUTION OF THIS AND HOW IT
CAME THAT WAY IS A FASCINATING
AREA OF INVESTIGATION THAT IS
NOT FULLY UNDERSTAND CURRENTLY.
AND THE ONES WE'RE PARTICULARLY
INTERESTED IN ARE THESE LINE
ELEMENTS.
THESE ARE LONG INTERSPERSED
NUCLEOTIDE ELEMENTS.
IT'S ESTIMATED DEPENDS ON HOW
YOU LOOK AT THE SEQUENCES IN THE
GENOME AS BEING REVISED ALL THE
TIME, THAT THERE ARE AT LEAST
150 ACTIVE ELEMENTS, THAT MEANS
FULL LENGTH ELEMENTS.
AND IN HUMANS, AND OVER 3,000 IN
MICE.
SO WHAT IS THE MECHANISM JUST TO
PUT THIS IN VERY GENERAL TERMS
WHAT THE THINKING IS AB HOW THEY
FUNCTION, THERE ARE 6.2 KB, THE
FULL LENGTH ACTIVE ELM, A FIVE
PRIME PROMOTER UNTRANSLATED
ORF-1 WHICH IS A RNA BIND
PROGRESS TEEN AMONG OTHER THINGS
THAT IS THE -- IS ORF 1, ORF 2
CONTAINS ENDONUCLEASE AN REVERSE
TRANSCRIPTASE.
INTERESTINGLY THIS TRANSCRIBES
THE RNA AND THE TRANSLATED
PROTEINS THEN BIND BACK ON TO
THE RNA AND ORF-1 ACTING AS A
RNA BINDING PROTEIN, ALSO
RECRUITS IN THE ENDONUCLEASE AND
REVERSE TRANSCRIPTASE.
WHEN THE CELL WAS UNDERGOING
DIVISION THIS RIBONUCLEAR
COMPLEX IS TRANSPORTED TO THE
NUCLEUS THROUGH NOT YET WELL
UNDERSTOOD MECHANISMS WHERE THE
NUCLEASE CAN NICK THE DNA AND
REVERSE TRANSCRIPTASE TURN IT IS
RNA BACK TO DNA AND THIS CAN
INSERT WITHIN THE GENOME.
NOW, REPEAT THE FACT THAT THIS
PROCESS IS MUCH -- AS MUCH AS
IT'S MOSTLY THOUGHT OF TO OCCUR
WHEN THE CELL IS UNDERGOING CELL
DIVISION, THERE'S SOME EVIDENCE
THAT IT CAN HAPPEN IN A
NON-DIVIDING CELL BUT MOST
EVIDENCE SPORES IT REQUIRES A
DIVIDING CELL FOR THIS TO OCCUR.
THAT'S JUST THE -- ANOTHER PIECE
OF THIS IS THIS MACHINERY CAN
ALSO BE USED ON LINES TO
REGULAR mRNA AND SIGN ELEMENTS
CAN USE THIS MACHINERY TO BE
HIJACK SOD THEY CAN BE INSERTED
IN THE CENTRAL NERVOUS SYSTEM AS
WELL SO THE LINE ELEMENT IS ONE
ELEMENT THAT HAS THIS CAPACITY
FOR REINSERTION.
SO WE TOOK ADVANTAGE OF A METHOD
OR TECHNIQUE OR MARKER SEQUENCE
DEVELOPED BY JOHN WARREN AND
(INDISCERNIBLE) LAB TO TAKE A
HUMAN ARTIFICIAL LINE ONE AND
PUT A GFP WITH AN INTRON FLANKED
BY ACCEPTOR SITES INTO THE THREE
PRIME END.
THE ADVANTAGE IS ONCE THE RNA IS
TRANSCRIBED THE INTRON IS
DELETED T RNA FUSES WHEN THE
COMPLEX IS MADE AND INSERTED YOU
GET GFP OFF OF THE DNA SEQUENCE
WHICH IS INSERTED BACK IN THE
GENOMES SO THIS TB USED AS A
MARKER NOT ONLY FOR WHETHER OR
NOT THIS IS EXPRESSED BUT
WHETHER OR NOT IT'S INSERTED IN
THE GENOME.
USING THIS MARKER WE TESTED OUR
NEURAL PROGENITOR CELLS IN
CULTURE.
WE COMPARE THIS TO FIBER BLAST,
MEZ CHIEMAL STEM CELLS AND OTHER
STEM CELLS AND THE OTHER ONES WE
SAW GFP EXPRESSING CELLS WERE IN
THE NEURAL PROGENITOR CELLS AND
ONLY WHEN WE INFECTED THEM AT
THE LAST STAGE OF THEIR CELL
DIVISION.
WHEN THEY WERE HIGHLY
PROLIFERATIVE IN THE NON-CCG
STATE OR MIX THE STEM CELL STATE
VERY LITTLE.
WE CAN PROVE THIS BY SHOWING THE
SMALLER PIECE, DELETED PIECE
EXISTS IN THE GENOME OF THESE
MPCs AT THIS STATE.
NOW, IN VITRO WE DID EXPERIMENTS
WITH IN VITRO AND WE WANTED TO
MAKE SURE THAT THE INSERTIONS
WERE -- THIS IS A PHENOMENON
WORTH HAPPENING, I SHOULD SAY WE
CONFIRMED THE MICROARRAY
EXPERIMENTS WITH PCR SHOWING IT
WAS ACTUALLY THESE CELLS DID
MAKE A LOT OF THE LINE SEQUENCES
THAT WE SAW.
IN FACT, QUITE SURPRISINGLY AT
THIS TWO DAY, THREE DAY STAGE
AFTER THE CELLS ARE BEGINNING TO
DISSESHIATE WITH THE NEURONS ONE
OF THE MOST ABUNDANT RNA
TRANSCRIPT ARE THE LINE
TRANSSCRICTS.
WHETHER THE TISSUE DULLTURE ARE
REAL, WE MADE A CONSTRUCT WITH
FLANKING AREA EXPRESSED.
ONE OF THE MOST GREAT FUND FOR
US WHEN WE TOOK THE ADULT BRAIN
TISSUE AND EXAMINED IT AND WE
SAW GREEN CELLS THROUGHOUT THE
CENTRAL NERVOUS SYSTEM IN THE
STRIATUM AND CEREBELLUM, HAD
BEAUTIFUL MORPHOLOGY OT CELLS.
AND IN AREAS BUT THEY WERE NOT
EXPRESSED IN ASTROCYTES IN OUR
HANDS OR IN MICROGLIA BUT ONLY
RATHER OR OLIGODENDROCYTES FOR
THAT MATTER BUT REALLY ONLY IN
NEURONS.
WE COULD DO LASER CAPTURE,
SINGLE CELL LASER CAPTURE AND
CONFIRM THESE GREEN CELLS HAD
INSERTIONS AS APPROPRIATELY
WHEREAS NEGATIVE CELLS IF YOU
JUST TAKE IT OUT THERE YOU
WOULDN'T SEE THIS.
SO SUGGESTING THESE WERE
AUTHENTIC INSERTIONS OCCURRING
IN VIVO.
WHERE DO THEY GO?
THIS IS EARLY WORK TRYING TO
ADDRESS THE QUERKS TWO MAIN
QUESTIONS IN THIS IS DOES THIS
REALLY OCCUR, WHERE DOES IT
OCCUR, AND WHAT IMPACT MIGHT IT
HAVE.
AND THIS WAS SOME OF THE EARLY
EFFORTS IN VITRO TO DO THAT
USING REPORTER CONSTRUCTS.
SO ONE OF THE THINGS THAT WE
COULD DO WITH THESE CLONES WAS
TO GROW UP CLONES INDIVIDUALLY
AND THEN USE INVERSE PCR USING
SEQUENCES WITHIN THE LINE
ELEMENT SEQUENCING OUT FROM THE
LINE ELEMENT AND WE COULD
IDENTIFY WHAT GENE WILL BLAST
THE SEQUENCES BACK TO THE GENOME
AND IDENTIFY WHERE THE LINE
INSERTED WITHIN THE GENOME OF
THAT CLONE OF CELLS.
WE HAVE DONE THIS FOR ABOUT 50
INDIVIDUAL CLONES AND A LOT OF
THE GENES ARE NEURONAL OR HAVE
NEURONAL FUNCTION REPORTED,
NEURONAL FUNCTION, SOME
HOUSEKEEPING AND CERTAINLY OTHER
GENES AS WELL.
THIS CAN BE ART FACTUAL BECAUSE
WE ARE GROWING THE CELLS AS
NEURAL PROGENITOR CELLS SO
LIKELY NEURAL RELATED GENES ARE
OPEN, THEIR CHROMATIN IS MORE
OPEN AT THAT STAGE, MAYBE THAT'S
A REASON WHY INSERTIONS OCCUR IN
THOSE AREAS.
THAT'S A CAVEAT IN THE
DESCRIPTION, DISCUSSION OF THAT.
CAN LINE INSERTION CHANGE GENE
EXPRESSION?
SO ONE OF THE WAYS THAT WE HAVE
DONE THIS WITH SEVERAL GENES IS
TO IDENTIFY WHERE THEY HAVE
INSERTED AND DETERMINE WHETHER
OR NOT THERE'S ANY PHENOTYPE.
HERE IS ONE EXAMPLE USING PSD 93
AS INSERTION SITE SO WE MAPPED
THE LINE JUMPED TO THE PROMOTING
5 PRIME IS PSD-93.
WE CHOSE THIS ONE TO TALK ABOUT
BECAUSE THIS CLONE CL-22 SHOWS
AN OVEREXPRESSION OF PST-93
UNDER NORMAL STEM CELL
CONDITIONS WHEREAS IN WHY WILD TYPE
IT'S NOT THERE.
BOTH RNA IN TERMS OF PROTEIN.
ALSO BECAUSE THIS CLONE WHEN WE
DIFFERENTIATE THESE CELLS WITH
RETINOIC ACID WHICH IS OUR
GENERAL WAY OF DIFFERENTIATING
CELLS WHEN WE PULL FGF AWAY,
NORMALLY THE WILD TYPE
POPULATION GIVES ABOUT 60%
NEURONS GLIA AND
UNDIFFERENTIATED OR UNIDENTIFIED
CELLS BUT THIS CLONE WITH THE
SAME CONDITION DIFFERENTIATED
MOSTLY INTO NEURONS.
SO TO EXAMINE THAT WE CLONED THE
RAT VERSION, THESE ARE RAT
CELLS, AND OVEREXPRESSED PFD-93
INTO THESE CELLS AND COULD
EVIDENCE THE FACT THEY SHOWED A
GREATER PROPENSITY FOR GIVING
RISE TO NEURONS.
AND THEN WHEN HE WE KNOCK DOWN
PST-93 IN THE CL-22 CLONE, WE
RESCUED SOME ELEMENTS OF THE
PHENOTYPE, LESS NEURONS SO
SUGGESTING THE FACT THAT ONE
INSERTION OR THIS PARTICULAR
INSERTION BY VIRTUE OF THIS
CLONE WAS HAVING SOME EFFECT ON
THE FATE OF THE CELLS.
CONTROLLING ALL OF IT BUT MIGHT
HAVE SOME CONTROL.
THIS IS A SMALL REDUCED SLIDE SO
HARD TO READ BUT THE POINT HERE
IS WHAT ARE THE CONSEQUENCES OF
INSERTION?
THE CONSEQUENCES CAN BE
MULTIPLE, THEY COULD INSERT IN A
CODING SEQUENCE INTERRUPTING THE
FUNCTION OF A GENE.
THEY CAN INSERT INTO NON-CODING
SEQUENCES WHICH WOULD RESULT
PERHAPS IN CHANGING SPLICE
VARIATION THEY CAN ADD POLYA
TAILS TO ELONGATE THE SIZE OF A
GENE, ONE INTERESTING FEATURE
THE LINE ELEMENT HAS INTERNAL
PROMOTERS THAT EXPRESS IN BOTH
DIRECTIONS SO THIS AFFORDS THE
OPPORTUNITY FOR SOME INTERESTING
MISCHIEF ON THE PART OF THESE
LINE ELEMENTS WITH JUST
NON-CODING SEQUENCES INDEPENDENT
OF WHETHER OR NOT THEY INSERT
AND THEN AS I'LL SHOW YOU LATER
THEY MIGHT HAVE SOME IMPACT ON
EPIGENETIC PHENOMENA THROUGH
THEIR IMPACTS ON METHYLATION.
SO EVERYTHING I HAVE TALKED
ABOUT SO FAR IS USING THIS
REPORTER CONSTRUCT AS A METHOD
OF LOOKING AT IT.
AND OF COURSE THAT HAS
ADVANTAGES BUT IT ALSO HAS
DISADVANTAGES.
ONE DISADVANTAGE, IT'S A SINGLE
REPORTER AND THE MICE THAT WE
HAVE BEEN LOOKING AT THERE ARE
3,000 ELEMENTS SO WE'RE JUST
LOOKING AT THE TIP OF THE
EXPERIMENT HERE.
WE WANTED TO DEVELOP METHODS TO
LOOK AT THE ENDOGENOUS LINE
ELEMENTS AND THE FIRST WAY THAT
WE CHOSE TO DO THAT WAS
DEVELOPED BY ANY COAL COFELL AND
CAROL MARCETA IN THE LAB.
THIS IS A RECENT ELABORATION ON
THAT METHOD BY MIKE MCCONNELL
WHERE HE TAKES INDIVIDUAL CELLS
AND USES AN ANTIBODY TO NEW ENDS
AND SORTS THE CELLS, PLACE IT IS
CELLS IN A 96 WELL PLATE AND
DOES THE SAME TISSUES FROM
HEART, LIVER AND OTHER TISSUES.
HERE IS THE THEORY IS THAT IF IN
FACT THERE ARE LINE ELEMENTS AND
THERE'S 3,000 OF THEM ACTIVE IN
THEY ARE JUMPING TO THE DNA INTO
THE GENOME THEN YOU MAYBE ABLE
TO PICK UP A COPY NUMBER
VARIANT, YOU MAYBE ABLE TO PICK
UP A CHANGE IN THE TOTAL AMOUNT
OF DNA IF YOU USE PRIMERS FOR
THE LINE ELEMENTS.
SPECIFICALLY AND COMPARE THAT
WITH SEQUENCES THAT MAYBE OF
HIGH ABUNDANCE BUT NOT MOBILE IN
THE THE DNA.
SO IN THIS CASE HE DID SINGLE
CELL 96 WELL PLATE USING QPCR
AND IN THE SAME WELL MIXING
PRIMERS FOR ORF-2 AND 5S AS WELL
AS SATELLITE AND OTHER DNA NOT
MOBILE.
FINDS THE THRESHOLD FOR BOTH AND
THEN CALCULATES A DECT BETWEEN
THE TWO W A LOWER NUMBER BEING
THE HIGHER AMOUNT OF DNA COPY.
THAT IS REQUIRED TO REACH
THRESHOLD.
DOING THIS ONE MIGHT FIND THAT
PLOTTING 80 CELLS OF THESE, TO
80 CELLS PLOTTED, CUMULATIVE
MOBILITY GRAPH, THEY ALL MAP
AROUND EACH OTHER AND THE HEART
AND HIPPOCAMPUS AND THE OTHER
NEURAL TISSUES THAT HE'S DONE
ALWAYS SHOW A GREATER AMOUNT OF
DNA CONTENT WITHIN THE GENOME OF
THESE INDIVIDUAL CELLS.
OBVIOUSLY THERE'S VARIABILITY
FROM THOSE NOT DIFFERENT TO
THOSE THAT ARE ACTUALLY QUITE
DIFFERENT.
AND THIS IS ALSO PILOT NEW DATA
THAT HE'S GENERATING USING THIS
NEW METHOD HERE PLOTTING 432
INDIVIDUAL NEURONS FROM THE
CORTEX OF THE FRONTAL CORTEX OF
ONE MOUSE AND COMPARING THAT TO
THE CORTEX OF A SECOND
GENETICALLY IDENTICAL AGE
MATCHED SEX MATCHED SO
MONOZYGOTIC TWIN OF THAT MOUSE
AND THESE TWO MICE HAVE
DIFFERENT NUMBERS OR DIFFERENT
AMOUNTS OF DNA IN THEIR
INDIVIDUAL CELLS.
SO NOT ONLY ARE THERE
DIFFERENCES BETWEEN BRAIN AND
OTHER ORGANS IN TERMS OF
INCREASES, BUT INDIVIDUAL CELLS
BETWEEN ANIMALS ARE DIFFERENT
FROM EACH OTHER.
SO THE OTHER FEATURE OF THIS IS
THAT WHEN LOOKING AT -- THIS IS
AN OLDER PLOT, THAT NEEDS TO BE
UPDATED BUT IT HOLDS STILL, THAT
AS NOT CORRECT TO PLOT IT IN
THIS WAY IN SOME LINEAR INCREASE
BUT THIS IS SIZE IN ANY EVENT,
THE NUMBER OF THE PERCENT OF THE
NON-CODING MOBILE ELEMENTS THAT
EXIST IN THE GENOME AND HUMAN
HAS ACTUALLY MORE THAN OTHER
SPECIES.
SO IT'S NOT GOING AWAY BUT
RATHER INCREASING FOR SOME
REASON.
THIS IS AN OLDER PAPER BY HAGUE
(INDISCERNIBLE) AND ALSO JOHN
(INDISCERNIBLE) IN THE STUDY
TRYING TO ESTIMATE HOW MANY
ACTIVE ELEMENTS EXISTED IN THE
HUMAN.
AND THE WAY THEY DID IT WAS TO
MAP FOR HUMAN A TRANSFORMED CELL
LINE THAT WOULD ACTIVELY ALLOW
FOR INSERTIONS TO TAKE PLACE.
THEY CHARACTERIZE HERE WHICH ARE
THE ELEMENTS ARE ACTIVE AND
WHICH ONES WERE LESS ACTIVE AND
PROPOSE THERE WERE A SMALLER
NUMBER MORE ACTIVE A SMALLER
NUMBER WERE HIGHLY ACTIVE.
DIFFICULT TO REDOO RETHINK ABOUT
THIS REMEMBERING THESE ARE PUT
INTO THE CONTEXT OF ANOTHER CELL
SO WE WOULD HAVE TO REVISE OUR
THINKING ABOUT THAT.
BUT IT GIVES US AN IDEA SO WE
WANTED TO SEE WHETHER OR NOT
THERE WAS LINE ELEMENT MOBILITY
IN HUMAN CELLS AND SINCE THE
LINE MOBILITY IS OCCURRING
PRIMARILY AT THIS EARLY STAGE WE
WANTED TO WATCH THAT PART OF IT
AS WELL.
SO ONE WAY TO DO THAT IS TO USE
HUMAN EMBRYONIC STEM CELLS, SO
WE HAVE DEVELOPED PROCEDURES
WHERE WE COULD TAKE HUMAN ES
CELLS, MAKE RO SETS AND
DIFFERENTIATE THE CELLS INTO A
NEUROPROGENITOR POOL.
THAT NEURAL PROGENITOR POOL?
OUR HANDS A REPRODUCIBLE GROUP
OF CELLS THAT WE CAN PROPAGATE,
FREEZE DOWN, THAW AND DO
EXPERIMENTS WITH.
SO WE CAN THEN TRANSIF HE CAN
THESE CELLS AND DIFFERENTIATE
THEM NOW IN OUR DIFFERENTIATING
CONDITION.
WHAT WE FOUND WAS USING A
REPORTER CONSTRUCT WITH A LINE
PROMOTER DRIVING LUCIFERASE
THERE WAS A DRAMATIC INCREASE IN
ACTIVITY AT TWO DAYS AT THE TIME
WHEN THE CELLS ARE JUST BECOMING
NEURONS AND IN LOOKING AT THE
NUMBER OF GREEN CELLS THAT WERE
THERE, WE DEFINITELY SAW A
SIGNIFICANT NUMBER OF CELLS THAT
SHOWED AN INCREASE IN GFP
POSITIVITY SUGGESTING THERE WAS
INSERTION.
WE FULLY DIFFERENTIATED THE
CELLS, WE WANT TO SEE WHETHER OR
NOT THIS INSERTION WAS LETHAL.
AND THE ONES THAT DID HAVE
INSERTIONS DIED.
BUT WE FOUND PLENTY OF GREEN
CELLS THAT WERE NEURONS THAT WE
COULD PATCH CLAMP, THEY HAD
ELECTROPHYSIOLOGICAL PROPERTIES
SO THE INSERTION BY ITSELF WAS
NOT LETHAL.
BUT IT DID OCCUR.
NOW IF IT DOES OCCUR IN THIS IN
VITRO SETTING COULD IT OCCUR IN
IN VIVO SETTING AND GIVEN THE
FACT MIKE AND NICOLE AND CAROL
DEVELOPED THIS TECHNIQUE FOR
MOUSE SINGLE CELL WE TRIED TO DO
SINGLE CELL PCR FOR HUMAN
AUTOPSY MATERIAL, WE HAVEN'T
BEEN ABLE TO GET THAT DONE YET,
WE CAN TAKE CHUNKS OF POSTMORTEM
TISSUE AND FROM THE SAME
PATIENTS WE GET A VARIETY OF
TISSUES AND WE CAN GET HEART AND
LIVER TISSUE FROM THE PATIENTS.
SO WE MAKE GENOMIC DNA AND DO A
SIMILAR QPCR MULTI-PLEXING
REACTION, NOW LOADING WITH ABOUT
80 PICO GRAMS OF STARTING
MATERIAL FOR THE REACTION THE
TAKE PLACE.
NOW, THE THEORY IS THAT IF THERE
ARE MORE'S THERE'S JUMPING OR
INSERTION OF LINE ELEMENTS TO
THE GENOME OF THE HUMAN BRAIN WE
SHOULD SEE A GREATER AMOUNT OF
DNA CONTENT RELATIVE TO OTHER
TISSUES.
I WOULDN'T TELL YOU THIS IF IT
WEREN'T TRUE.
WE HAVE SEEN IN ALL CASES A
GREATER AMOUNT OF DNA CONTENT
THAN DID THE LIVER, HEART OR
OTHER TISSUES.
AND WE USE THESE COMPARED TO TWO
DIFFERENT NON-JUMPING NON-MOBILE
ELEMENTS AS CONTROLS WE SEE NO
DIFFERENCE BETWEEN THEM.
IT LOOKS LIKE THIS IS ACTUALLY A
QUITE REASONABLE.
BUT AS YOU SAW, WE SAW SOME
DIFFERENCE BETWEEN THE
CEREBELLUM AN HIPPOCAMPUS SO WE
TOOK THIS FURTHER AND STARTED
DOING MICRODISSECTIONS OF HUMAN
BRAIN TISSUE TO SEE WHETHER OR
NOT THERE WAS A PATTERN THAT
EMERGED, ANTICIPATING MAYBE THE
MOBILE -- THE AREAS OF GREATEST
MOBILITY MIGHT BE ASSOCIATED
WITH THESE PROLIFERATIVE ZONES,
THAT DIDN'T QUITE HOLD UP THOUGH
WE'RE NOT FINISHED WITH THIS
ANALYSIS.
BUT JUST HAVE IT SAID THAT THERE
ARE SIGNIFICANT DIFFERENCES
BETWEEN AREAS OF THE BRAIN FROM
EACH OTHER THAT WE'RE TRYING TO
UNDERSTAND, AND THE VARIANCES
SMALL ENOUGH SO IT LOOKS
BELIEVABLE.
IF IT WERE RANDOM AND JUMPING
EVERYWHERE, SO ONE THING WE
WANTEDDED TO DO IS GUESS AT HOW
MANY INSERTIONS OCCUR IN HUMAN.
IF YOU HAVE A TOTAL OF 150
ACTIVE ELEMENTS AND MAYBE A
SMALLER NUMBER OF THAT BEING
ACTIVE AT ANY ONE TIME, HOW MANY
SUCCESSFUL INSERTIONS WOULD YOU
SEE?
THERE'S LOTS OF RNA MADE SO
POTENTIAL FOR ONE LINE ELEMENT
TO INSERT IS HIGH.
SO WE DID THIS EXPERIMENT THIS
WAY, IT REQUIRE AS LITTLE BIT OF
CALCULATION AND EXPLANATION FOR
THE BACK OF THE ENVELOPE METHOD,
WE WENT BACK TO THE HIPPOCAMPUS
AND LIVER WHERE WE GOT A NICE
DIFFERENCE BETWEEN THE TWO THERE
USING THIS RATIO METHOD AND WE
SPIKED BACK INTO THE LIVER
COPIES OF LINE SEQUENCE.
SO LINE PLASMIDS SO TEN, 100,
1,000 OR TEN THOUSAND.
AND THEN RAN THE REACTION WITH
LIVER TO SEE HOW MANY EXTRA
PLASMIDS IT WOULD TAKE TO COME
TO THE DNA CONTENT SIZE OF THE
WILD TYPE HIPPOCAMPUS.
AND SOME PLACE BETWEEN 10,000,
1,000 AND 10,000 PLASMID PER
80,000 OR 12 CELLS, SO IF YOU
FIGURE THAT OUR STARTING SAMPLE
WAS 80 PIE CO-GRAMS OF DNA AND
EACH GENOME IS 6.6 PIE CO-GRAMS
OF DNA THAT'S 12 CELLS
APPROXIMATELY DIVIDED BY THESE
NUMBERS.
GIVES YOU BACK OF THE ENVELOPE
CALCULATION OF THERE BEING
SOMEWHERE BETWEEN 80 AND 300
INSERTIONS OF LINE SEQUENCE IN
EVERY CELL ON AVERAGE, THAT
SURVIVE INTO ADULT.
OR THIS GROUP OF PATIENTS.
SO HOW IS IT REGULATED?
THIS IS JUST A SUMMARY OF A
STUDY PUBLISHED RECENTLY WHERE
WE'VE LOOKED AT THE LINE
SEQUENCE AND FOUND THAT THERE'S
A VARIETY OF INTERESTING DNA
BINDING DOMAINS, MOST
INTERESTING FROM OUR PERSPECTIVE
IS THERE'S A SOX-2 BEHINDING
DOMAIN WHICH PLAY AS ROLE AS IT
TURNS OUT IN SUPPRESSING LINE
ACTIVITY.
SO SOX2 ACTS AS SUPPRESSOR THAT
CONTAINS OTHER MOLECULES.
WHEN THE CELLS DIFFERENTIATE TO
OTHER NEURONS SOX2 COMES OFF AND
CELLS ARE ACTIVATED BY WENT 3A
THROUGH BETA CATENIN THROUGH TCF
TO BIND TO THE DNA OF THIS LINE
ELEMENT AND THAT INDUCES
ACTIVITY OF THE LINE DURING THIS
PERIOD OF TIME WHEN CELLS ARE
UNDERGOING DIFFERENTIATION.
SOMEKNISTICLY IT LOOKS AS THOUGH
THIS MACHINERY IS QUITE
IMPORTANT FOR THE ACTIVATION OF
THE LINE IN NEURAL PROGENITOR
CELLS.
INTERESTINGLY THESE ARE THE SAME
FACTORS THAT ARE USED BY GENE Z
SUCH AS NEUROD-1, A NEURAL
PROGENITOR FACTOR,
DIFFERENTIATION FACTOR AND THEY
USE THE SAME MACHINERY.
SO THERE'S A REDUNDANCY OF
TRANSCRIPTION MACHINERY BEING
USED TO ACTIVATE THE LINE AS IT
IS TO ACTIVATE NEURONS.
IN ADDITION TO THE SOX2 BINDING
SITE WHEN WE LOOKED AROUND THE
SOX2 SITE WE FIND THAT THEY'RE
SURROUNDED BY ISLANDS OF CPG OR
METHYLATION SITES.
WE LOOK AT THAT A LITTLE BIT
MORE CAREFULLY TO REMIND YOU CPG
ISLANDS OR BINDING SITES ARE
SITES FOR METHYL BINDING
PROTEINS OF WHICH THERE ARE A
VARIETY THAT ARE ACTIVELY ACTING
AS SUPPRESSORS AND CURRENT
WORKING HYPOTHESIS ABOUT THIS IS
THAT THEY'RE GLOBAL REPRESSORS
THOUGH OBVIOUSLY THERE'S
EVIDENCE THAT REDUCTION IN
MECP-2 OR DELETION OF THESE
EPIGENETIC MECHANISMS WILL
RESULT IN UP REGULATION OF SOME
GENES.
IN ANY EVENT THEY'RE GLOBAL
REPRESSORS IN GENERAL AND THE
DISRUPTION OF MECP-2 ACTIVATES A
VARIETY OF GENES NORMALLY UNDER
SUPPRESSED CONDITIONS.
SO GIVEN WE FOUND THESE MECP-2
SITES WHICH WE KNOW TO BE AN
IMPORTANT SUPPRESSOR OF LIME 1
WE DECIDE TO SEE IF MECP-2 PLAYS
A ROLE IN ACTIVATION OF LINE
ELEMENT IN NEURAL PROGENITOR
POPULATION.
SO IF YOU USE METHYLATION TO
INCREASE METHYLATION YOU CAN GET
A SUPPRESSION OF LINE ACTIVITY
USING IN THIS CASE LUCIFERASE
CONSTRUCT WHICH HAD THE LINE-1
UTR BEING DRIVEN SO LOOKS LIKE
METHYLATION CAN HAVE AN EFFECT
ON TRANSCRIPTION WE ALSO SHOW
THAT MECP-2 IS BOUND TO THIS
FIVE PRIME UTR REGION ITSELF AND
WHEN CELLS DIVIDE THERE IS LESS
BINDING OF MECP 2 TO THE DNA OF
THAT PROMOTER.
THIS ALSO CAN BE ALLEVIATED BY
5A SITODENE.
SO IN THIS POINT WE MUS TO THE
MECP-  MOUSE.
THE MOUSE THAT WAS GENERATED
DELETED ACTIVE FORM OF MECP-2 SO
IT NO LONGER ACTS AS A GLOBAL
SUPPRESSOR.
THERE'S A VARIETY OF MICE OUT
THERE ONE CAN USE.
THE LUCIFERASE CONSTRUCT IN
THESE CELLS, SAW A DRAMATIC
INCREASE IN ACTIVITY OF THE LINE
PROMOTER IN THESE CELLS.
WE THINK THAT'S MECP-2 DEPENDENT
BECAUSE IF WE OVEREXPRESS MECP
MANUFACTURE 2 IN THAT LINE WE
CAN SUPPRESS IT BACK TO THE WILD
TYPE LEVEL SUGGESTING THIS IS A
REGULATOR, WE SEE THIS IS ALSO
TRUE FOR OUR TWO AGAIN
SUPPORTING THE IDEA IT'S IN
BOLD.
SO TO TEST THIS IN VIVO WHAT WE
DID WAS TO TAKE OUR REPORTER
MOUSE WHICH HAD THE LINE EGFP
AND CROSSED THESE INTO THE
MECP-2 MINUS MOUSE.
OUR PREDICTION HERE IS THAT THE
LACK OF LINE SUPPRESSION WOULD
RESULT IN A GREATER AMOUNT OF
GFP POSITIVITY IN THE CELLS.
AND IT WAS REALLY VISUALLY
STRIKING AS YOU GO THROUGH
RAREIOUS AREAS OF THE BRAIN, I
DON'T KNOW THAT YOU CAN SEE THIS
BUT HERE IS CEREBELLUM IN THE
PAST WE WOULD SEE INDIVIDUAL
CELLS, NOW WE SEE CLUSTERS OF
CELLS LINEAGE, A LARGE NUMBER OF
CELLS WITHIN CERTAIN AREAS.
WE HAVE GONE AND MAPPED OUT IN A
VARIETY OF THESE ANIMALS TO SEE
WHERE THE INCREASE IN GFP OCCURS
OR WHERE THE INSERTIONS ARE
OCCURRING.
IT'S NOT EVERYWHERE, THERE ARE
CERTAIN AREAS OF THE BRAIN WHERE
YOU SEE A SIGNIFICANT AMOUNT OF
INCREASE, ANOTHER, NOT SO MUCH.
WE DEVELOP SOME TOOLS, SOME
QUANTITATIVE TOOLS TO TRY TO GET
A BETTER SENSE OF THE THREE
DIMENSIONAL PEER SPECKTIVE WHERE
THEY GO -- PERSPECTIVE WHERE
THEY GO.
WE D A SERIAL SECTION AND MOUNT
THEM AND THEN RECONSTRUCT AN
ENTIRE BRAIN AND MAP OUT WHERE
THEY GO.
WHAT WE FIND IS THAT THE
STRIATUM AN CEREBELLUM WE GET
TWO TO SIX TIMES AS MANY
INSERTIONS AND THEY'RE OFTEN IN
CLUSTERS OF CELLS.
SO A SIGNIFICANT NUMBER OF CELLS
OCCURRING.
THE INSERTIONS ARE OCCURRING AT
ALL TIMES WHEN THE CELLS ARE
UNDERGOING CELL DIVISION.
THE FIRST CELL DIVISION OR
NEUROGENESIS THAT OCCURS ARE E-9
TO E-11.
WE SEE IN THOSE AREAS WHERE
NEUROGENESIS JUST FIRST
HAPPENING WE SEE CELLS THERE.
AT E-13, HIGH CLUSTERS OF CELLS
THAT ULTIMATELY GENERATED CELLS
IN THE ADULT BRAIN BORN HERE,
THEY'RE GREEN AS WELL AS AN AREA
WHERE WE HAVE POSTNATAL
NEUROGENESIS LIKE THE OLT
FACTORY BULB IN THE HIPPOCAMPUS.
SO THIS PROCESS OF INSERTION IS
OCCURRING DURING THE EARLY STAGE
OF NEUROGENESIS BUT BOTH ALL
STAGES OF THIS EARLY STAGE OF
NEUROGENESIS BOTH PRE-NATALLY AN
POSTNATALLY.
THIS IS AGAIN ALL THE REPORTER
CONSTRUCTS SO WE WANTED TO THINK
ABOUT SEEING WHETHER WE CAN
DETECT IN THE DNA CONTENT OF AN
MECP-2 MOUSE VERSUS A WILD TYPE
MOUSE.
SO IN THIS CASE WE TOOK
INDIVIDUAL CELLS, SORTED THEM
AGAIN, AND PLATED THEM FROM WILD
TYPE VERSUS MECP-2, WE DO FIX
THEM IN SYNCHRONIZE THEIR CELL
CYCLE SO YES NOT BIASED BY THAT.
THEN AMPLIFY IN 96 WELL PLATES.
SO HERE WE'RE USING PRIMERS
WITHIN ORF-2 AND WE AND WE CAN
DETECT A SIGNIFICANT INCREASE IN
LINE DNA CONTENT WITHIN THE
MECP-2 KNOCK OUT RELATIVE TO
WILD TYPE.
WE HAVE DONE A SIGNIFICANT
NUMBER OF CONTROLS IN THE FIVE
PRIME UTR WHERE THERE'S NOT
JUMPING, TRUNCATED FOR THE MOST
PART, WE DONE SEE DIFFERENCES
BUT ALSO IN TO THE A LESS EXTENT
AND ALSO OUTSIDE THE LINE
SEQUENCES AN EVEN IN FIBROBLASTS
IF WE TAKE FIBROBLASTS AND LOOK
IN THE ORF-2 REGION WE DON'T SEE
A CHANGE IN MECP-2 SO THERE'S
DIFFERENCES IN THEIR MOBILITY,
CELL TYPE SPECIFIC MANNER.
SO GIVEN THAT THERE IS AN
INCREASE IN MECP-2 INCREASE IN
LINE INSERTION MECP-2 IN MICE WE
WANT TO LOOK AT THIS IN HUMANS
AS WELL.
SOME MAYBE FAMILIAR WITH THE
FACT MECP-2 MUTATION CORRESPONDS
TO A HUMAN SYNDROME CALLED RET
SYNDROME WHICH IS PART OF THE
LARGER SYNDROME OF AUTISTIC
BEHAVIORS, A DEVELOPMENTAL
DISORDER THAT STARTS NORMAL BUT
LEADS TO CNS DEFICITS WHICH ARE
AS I SAID, RELATED TO AUTISM.
BUT PROBABLY A DISTINCT SYNDROME
IN AND OF ITSELF.
WE ALREADY KNEW SOMETHING ABOUT
MORPHOLOGY THAT HIPT DIFFERENCES
IN THE CELLS SO THEY HAVE CELLS
IN THE BRAIN SO NORMAL NEURONS
COMPARED TO RATS IN AUTISM
CELLS, THE AUTISM CELLS ARE
GENERALLY SMALLER, THEY HAVE
FEWER BRANCHES AND HAVE SHOWN
THEY HAVE FEWER SYNAPTIC
CONNECTIVITY.
WE WANTED TO LOOK AT THIS IN
HUMAN BRAIN TISSUE.
WE HAVE HUMAN IPS TECHNOLOGY, WE
OBTAINED FIBROBLASTS FROM
CHILDREN AND SOME ADULTS.
WITH RET SYNDROME.
FIBROBLASTS, GREW THE FIBROBLAST
UP, OVEREXPRESSED THE FOURTH
TRANSCRIPTION FACTORS CAPABLE OF
REPROGRAMMING THE FIBROBLAST
INTO IPS CELLS.
THE OBJECTIVE WAS TO ADDRESS
THOSE IN NEUROPROGENITOR
POPULATIONS AND DETECT NEURONS
AND SEE IF WE CAN DETECT AN
INCREASE IN MOBILITY AT THAT
CRITICAL POINT.
SO WE DEVELOPED TECHNIQUES FOR
DOING THIS WHERE WE CANNOT ONLY
WITH ES CELLS BUT ALSO IPS CELLS
AND IN OUR HANDS THE IPS CELLS
BEHAVE VERY MUCH LIKE THESE LIKE
NORMAL ES CELLS, WE CAN CONVERT
THEM AND GIVE RISE TO NEURONS.
AT THIS POINT WE TAKE THE CELLS
AND TRANSFECT THEM AND MEASURE
ELECTROPORATION.
VERY CLEARLY IN THE RED CELLS WE
GET A GREATER NUMBER OF CELLS
THAT ARE GFP POSITIVE IN THESE
IPSC CELLS THAT ARE
DIFFERENTIATED INTO NEURONS.
REPEATEDLY IN OUR HANDS.
SO IN THE FINAL EXPERIMENT WHAT
WE WANTED TO DO WAS TO LOOK AT
HUMAN TISSUE, IF THEY ARE
SURVIVING WE HAVE GONE ON
ACTUALLY TO STUDY THESE CELLS IN
MORE DEPTH ONCE THEY
DIFFERENTIATED INTO NEURONS AND
ARE GFP POSITIVE AND WE DO, IT'S
INTERESTING WE SEE PHENOTYPES IN
VITRO OF THE CELLS BOTH THOSE
THAT A MARK FOR GFP AND THERE
ARE OTHER LINE ITEMS THAT ARE
ACTIVE OTHER THAN THESE.
THESE CELLS RECAPITULATE SOME OF
THE PHENOTYPES THAT WE HAVE SEEN
-- THAT WERE SEEN IN MOUSE AND
IN PATHOLOGY TISSUE, DECREASE IN
CELL SIZE AND SYNAPSES.
WE WANTED TO LOOK AT AUTHENTIC
TISSUE SO WE WERE FORTUNATE
ENOUGH TO GET TISSUE BANKS WHICH
CONTAINED TISSUE FROM HEART AND
OTHER TISSUES OF RET'S PATIENTS
THAT HAD PASSED AWAY.
AND COMPARED TO AGE MATCHED SEX
MATCHED CONTROLS.
THEN WE WENT BACK TO OUR METHOD
OF DETERMINING DNA IN THE GENOME
BY USING A QUANTITATIVE
MULTI-PLEKING TEXT MAN
METHODOLOGY.
AND AGAIN, THE THE THEORY BEING
THAT NOT ONLY WOULD WE SEE
DIFFERENCES BETWEEN HEART AND
BRAIN BUT WE WOULD SEE
DIFFERENCES IN THE NUMBER OF THE
DNA CONTENT BETWEEN THE TWO
BRAINS.
SO THESE ARE THE RESULTS THAT WE
SAW.
FIRST THING IS WE WERE ABLE TO
REPEAT THE DIFFERENCE IN HEART
BRAIN VERSUS HEART GREATER DNA
CONTENT.
AND BETWEEN THE WILD TYPE AND
RETES WE ALSO SAW A SIGNIFICANT
DIFFERENCE IN THE NUMBER OF DNA
COPIES THAT WE FINE IN THE
GENOME.
BUT NO SIGNIFICANT DIFFERENCE,
THIS IS TRENDING AND IT'S AN
INTERESTING PHENOMENA THAT WE
ARE CONTINUING TO EXAMINE
LOOKING AT OTHER TISSUES.
NEVERTHELESS THERE IS A
DIFFERENCE BETWEEN THESE TWO.
ALL OF OUR OTHER CONTROLS WE SEE
NO DIFFERENCE BETWEEN THE OTHERS
UNLESS WE USE OTHER CONTROLS
LIKE ORF-2 VERSUS THE FIVE PRIME
UTR, WE SEE THE SAME STATISTICAL
DIFFERENCE THAT WE SEE FROM
ORF-2 VERSUS SATE LIGHT.
-- SATELLITE.
SO SUMMARIZING, WHAT ARE WE
THINKING ABOUT?
WHAT IS THIS GUY TALKING ABOUT?
SO WE THINK THAT UNDER NORMAL
CONDITIONS THE LINES THAT ARE
TAKING -- EXIST WITHIN THE
GENOME UNDER NORMAL CONDITIONS
SUPPRESSED BY TRANSCRIPTIONAL
REPRESSING, BY EPIGENETIC
MECHANISMS OF SUPPRESSION, AND
THAT THEY'RE NOT ACTIVE IN
NORMAL SOMATIC CELLS BUT ONLY
WHEN THE CELLS OF NEURAL ORIGIN
WHICH HAVE THIS MACHINERY FOR
ACTIVATING LINE ELEMENTS AND
JUST AS THEY'RE UNDERGOING CELL
DIVISION, THEY'RE OPENED AND
LINE IS DRAWN AT THAT TIME.
AT THIS STAGE THESE CELLS WHEN
THEY HAVE THE APPROPRIATE
ENDENUCLEASE SITE THEY CAN
INSERT AND HAVE SOME IMPACT ON
THE CELL.
STOCHASTICALLY.
SO THE FINAL VIEW REALLY IS THAT
THERE ARE A VARIETY OF TIMES
THESE CELLS ARE ACTIVE.
IT'S BEEN SUGGESTED BY OTHERS
THAT AT THE GERM LINE CELL LINE
CAN BE ACTIVE.
AT ZYGOAT ACTIVITY THERE COULD
BE SOME ACTIVITY, THESE WOULD BE
GERM LINE CHANGES INTRODUCED BY
INSERTIONS OF LINE ELEMENTS.
NOW WE KNOW THAT EVEN AT THE
STILL CELL LAYER, EARLY STEM
CELLS WE HAVE SEEN SOME SMALL
AMOUNT OF INSERTION OCCURRING
BUT PREDOMINANTLY OCCURRING AT
NEUROGENESIS PERIODS, EARLY
DEVELOPMENT AS WELL AS LATE
STAGE DEVELOPMENT.
SO IN ADDITION MORE AND MORE
EVIDENCE SUGGESTING THAT THE
ACTIVITY OF THESE LINE ELEMENTS
CAN BE INFLUENCED BY A VARIETY
OF EXTERNAL FORCES DURING THESE
PERIODS OF NEUROGENESIS.
SOMETHING I CAN'T -- I HAVEN'T
GONE INTO IN MUCH DETAIL.
THE THEORY WE'RE WORKING ON IS
THIS MECHANISM IS ONE FOR
GENERATING A STOCHASTIC
DIVERSITY IN NEURONS THE THEORY
BEING THIS ADDING TO -- THIS
ADDS TO THE SEMATIC NEUROLOGICAL
DIVERSITY BETWEEN INDIVIDUALS
AND MODIFICATIONS IN THIS
CONONCAL AND HOMEOSTATIC
MECHANISM FOR GENERATING
DIVERSITY CAN IN AND OF ITSELF
CONTRIBUTE TO DISEASE AS IN THE
CASE THAT WE WOULD SPECULATE IN
THE CASE OF RET SYNDROME.
BUT AT A MINIMUM DIVERSITY IS
ADDING TO BEHAVIORAL DIVERSITY
OR INDIVIDUAL DIFFERENCES.
AND RATHER EXTREME HYPOTHESIS
WOULD BE THAT SOME BEHAVIORAL
DIVERSITY THAT EXISTS IN A
MAMMALIAN POPULATION IS
ATTRIBUTABLE TO THIS SEMATIC
EVOLUTION OR SEMATIC MOW SAFE
HARBORRISM THAT'S DERIVED IN THE
BRAIN AND THAT ONE CAN EXPAND ON
THAT BY INCREASES MOBILITY, OR
IF ONE WERE TO REDUCE OR
ELIMINATE LINE MOBILITY IN THESE
EARLY STAGES THE MEAN BEHAVIORS
OF INDIVIDUALS WOULDN'T CHANGE
BUT THE VARIANTS OR THE
DIFFERENCES BETWEEN PEOPLE WOULD
BE DIFFERENT.
SO I HOPE I HAVE BEEN A LITTLE
PROVOCATIVE HERE, MAYBE NOT TUCH
SUGGESTING LINE SEQUENCES
FORMING GENETIC MOSAICISM IN THE
BRAIN AN THIS IS ACTUALLY A
BEGINNING OF A VARIETY OF OTHER
MOBILE ELEMENTS THAT WE MIGHT
SEE ACTIVE.
WE'RE WORKING ACTIVELY RIGHT NOW
ON ALLOS AND THERE'S GOOD
EVIDENCE FOR THE FACT THEY'RE
MOBILE IN SEMATIC TISSUE.
WE LEARNED SOMETHING ABOUT THE
REGULATION THERE IS AN
INTERESTING PARALLEL WHEEN THE
REGULATORY SEQUENCE AN ACTIVITY
OF LINES AND JEERN TRANSCRIPTION
MACHINERY INVOLVED IN MAKING
NEURONS IN THE BRAIN.
AND MOST PROVOCATIVELY WE THINK
THE TISSUE SPECIFIC GENETIC
VARIATION CHALLENGES THIS
CONCEPT OF A STATIC GENOME AS
BARBARA MCCLINTOCK SUGGESTED.
AND THIS HAS IMPLICATIONS WE
BELIEVE FOR GENETIC AND
INHERITED AND NON-INHERITED
DISEASE, THIS IS A SEMATIC
EVENT.
PHILOSOPHICAL PROPOSITION IS YOU
ARE UNIQUE, NOT ONLY ARE YOU A
PRODUCT OF YOUR GENES IN YOUR
ENVIRONMENT BUT ALSO A PRODUCT
OF CHANCE.
SO THESE ARE SOME OF THE PEOPLE
IN THE LAB THAT HAVE BEEN BRAVE
ENOUGH TO WORK ON THIS TOPIC
OVER THE LAST FEW YEARS, SOME
ORIGINALLY STARTED AND LEFT IN
DESPERATION.
OTHERS HAVE STUCK WIT AND SOME
HAVE EVEN GOTTEN JOBS AS A
CONSEQUENCE OF IT.
BUT WE'LL KEEP AT IT AND I THANK
YOU VERY MUCH FOR YOUR
ATTENTION.
[APPLAUSE]
DO I TAKE QUESTIONS?
OKAY.
I'M HAPPY TO TAKE SOME
QUESTIONS.
>> I ASKED A QUESTION OVER HERE,
THIS IS A VERY EXCITING TALK, I
WOULD LIKE BEFORE I ASK MY
QUESTION A POINT OF
CLARIFICATION OF YOUR REPORTER
VECTOR WITH REVERSE INTRON AND
GFP, IS THAT DRIVEN BY A
HETEROLOGOUS PROMOTER OR IS THAT
DRIVEN BY THE LINE PROMOTER?
>> THE LINE PROMOTER.
>> LINE PROMOTER.
I'M WONDERING ABOUT BEING ABLE
TO SEQUENCE INDIVIDUAL
INTEGRATION EVENTS AND THEIR
POSITIONING IN THIS NEUROLOGICAL
TISSUE.
AND YOU -- I WAS STRUCK BY BEING
ABLE TO SEE 80 TO 300 COPIES IN
THE HIPPOCAMPUS TISSUE.
CAN YOU USE THESE HIGH THROUGH
PUT SEQUENCING AND MEDIATED PCR
AND PULL OUT THESE --
INTEGRATION EVENTS?
>> I WAS GOING TO PRESENT SOME
OF THIS DATA WE HAVE BEEN
WORKING ON FOR THE LAST FEW
YEARS, I'LL TELL YOU A LITTLE
BIT ABOUT IT.
SO WHAT WE HAVE DONE AS YOU HAVE
SUGGESTED PAIRED IN SEQUENCING
AND USING PRIMERS WITHIN THE
THREE PRIME END THAT ARE
REASONABLY CANONCAL, THEY
CAPTURE ABOUT 30%, YOU CAN6ã2okw3z]okç
ç FINDçxyñ
SEQUENCES THAT ARE CONSERVE
REASONABLY CONSERVED AND DROP
THE DNA AND PUT ADAPTORS,
SEQUENCE IN BOTH DIRECTIONS AND
WE BLASTED THIS TO QUOTE UNQUOTE
THE GENOME OR THE GENOMES AND WE
DO SEE SIGNIFICANT NUMBER OF NEW
LINE ELEMENTS.
I KNOW OTHER PEOPLE ARE DOING
THIS AS WELL AND WE'RE COMPARING
NOTES ON THIS.
OUR THE ANALYSIS WHERE WE ARE
RIGHT NOW IS DETERMINING -- VERY
DIFFICULT TO GET CONFIRMATION ON
INSERTIONS SINCE IT'S OCCURRING
RANDOMLY IN EVERY CELL.
SO WE HAVE TO IN DNA AMPLY
KAYCATION AND GO BACK AND TEST
WHETHER OR NOT THE HITS WE FINE
ARE FOUND IN THAT TISSUE.
WE'RE IN THE COURSE OF DOING
THAT RIGHT NOW.
BUT THE BIG QUESTION IS ARE --
WE ARE SEEING WHAT APPEAR TO BE
-- WHAT WE'RE CALLING HOT SPOTS
OR AREAS WE'RE FINING IN THE
GENOME WE'RE FINDING MULTIPLE
COPIES OF INSERTIONS BUT WE'RE
ALSO FINDING LOTS OF PAIRED
COPIES OR INDIVIDUAL COPIES
THERE AS WELL.
AND DETERMINING WHETHER OR NOT
THAT'S REAL OR IS THAT SINGLE
COPY, IS THAT ART FACTUAL?
THESE ARE EARLY DAYS.
OTHER PART I CAN TELL YOU IS WE
ARE SEEING INSERTIONS IN OTHER
TISSUES, MORE THAN WE HAD
ANTICIPATED SO WE'RE ALL MY
STORY WAS TELLING YOU UNIQUE TO
THE BRAIN, WE CLEARLY ARE SEEING
NOW IN OTHER TISSUES WHAT WOULD
APPEAR NOVEL INSERTIONS.
THAT IS THE MAKING US WANT THE
GO BACK TO DATA TO MAKE SURE WE
HAVEN'T GOT ARTIFACT IN THERE.
BUT THAT'S -- YOU HAVE GOT IT
WHERE WE ARE RIGHT NOW.
>> ARE YOU TAKING ADVANTAGE OF
TONY FERANA'S VIEW WHICH THREE
PRIME ENDS ARE ACTIVE FOR
TRANSPOSITION?
ARE YOU SEE HOPPING OF THE
ACTIVE COPY?
>> WE'RE LOOKING SPECIFICALLY
FOR THE ACTIVE COPIES.
THAT WAS PART OF THE ORIGINAL
SOURCE WAS TO DO THAT.
OTHER PEOPLE THINKING TOTAL --
THERE'S A VARIETY OF WAYS THIS
IS BEING APPROACHED.
HAS A LOT TO DO WITH STARTING
MATERIAL THAT YOU'RE USING.
WE BELIEVE THIS IS ALL OCCURRING
UNIQUELY IN EACH INDIVIDUAL
CELL, THEY ARE LIKELY DIFFERENT.
SO OUR REAL AIM IS TO DO SINGLE
CELL ANALYSIS TO SORT OUT
INDIVIDUAL NEURONS TO AMPLY
AMPLIFY CASES OF THOSE
DIFFERENCES BETWEEN THEM.
>> WOULD A PREDICTION NOT BE IF
THE CELLS SURVIVE THAT IN THE
CASE OF RET SYNDROME YOU WOULD
END UP WITH MORE NEURONS IN THE
BRAIN?
>> IF -- NEURONS -- WHY WOULD
YOU SEE --
>> I THOUGHT YOU FOUND A
CORRELATION WITH YOUR LINE WITH
A NUMBER OF -- YOU GOT I THOUGHT
MORE PROGENITORS IN THE PICTURES
YOU SHOWED IN THE MICE.
WAS THAT CORRECT?
>> WE SAW -- NO, NO.
I'M SORRY.
YEAH.
SO WE DIDN'T SEE MORE NEURONS WE
SAW MORE GREEN NEURONS, THAT
MEANS THAT IN FACT THE BRAINS
ARE SOMEWHAT SMALLER SO WE
DIDN'T EVEN MAKE THAT CORRECTION
POINT BUT THERE ARE MORE CELL
THAT HAD LINE ACTIVITY IN THEM
BUT BRAIN SIZE IF ANYTHING IS
SLIGHTLY SMALLER IN THE RET
MOUSE.
I'M SORRY I DIDN'T MAKE THAT
CLEAR.
>> COULD THIS BE A MECHANISM FOR
IN MONOZYGOTIC TWINS IN BRAIN
SEQUENCE?
>> WE WOULD SPECULATE THAT,
WE'RE DOING THAT EXPERIMENT NOW
LOOKING AT MONOZYGOTIC TWIN
TISSUES BUT WE DO SEE THIS, THAT
WAS PART OF THE REASON WHY I
SHOWED YOU THE MONOZYGOTIC MICE.
THESE ARE INBRED MICE IN TAKING
INDIVIDUAL CELLS FROM SAME
REGION OF THE BRAIN SHOWING THE
AMOUNT OF INSERTION IS
DIFFERENT.
SO I WOULD AGREE WITH THAT.
THAT'S OUR FIRST ATTEMPT USING
MONOZYGOTIC MICE.
>> WOULD YOU EXPECT THEN WITH
ALL THIS DEBATE ON CLONING, IF
SOMEBODY WERE TO CLONE
THEMSELVES, THEY WOULD MAYBE
CROW UP WITH DIFFERENT PERSONAL
PERSONALITIES BASED ON THIS,
RIGHT?
WE
>> WE'RE A LITTLE EARLY FOR
THAT.
BUT THANK YOU.
YEAH.
>> COULD YOU SPECULATE ON THE
ADVANTAGE THAT NEURONS HAVE
BECAUSE OF THIS TRANSITION?
WHY NOT IN HEART AND CELLS AN
LIVER, WHAT KEEPS THEM IN CHECK
IN THOSE CELL TYPES?
>> SO WE ADDRESS THAT A LITTLE
BIT BY TAKING OTHER TISSUES AND
DID BISULFATE SEQUENCING AROUND
THE LINE PROMOTER.
IN THE LINE PROMOTER IN THE CPG
SITES OF FIBROBLASTS AND OTHER
TISSUES
CELLS.
THROUGH BY SULFATE SEQUENCING
LOOKS LIKE OTHER TISSUES ARE
MORE HIGHLY METHYLATED ON THE
PROMOTER THAN IN NEURONS SO THE
NEURONS SEEM LESS SIGNIFICANTLY
LESS METHYLATED IN THOSE
REGIONS.
THAT'S A CORELATION BUT THAT IS
IN FACT ONE THING WE DID SO WHY
WOULD THIS BE -- WHY -- TWO
PARTS OF YOUR QUESTION, WHY IS
IT UNIQUELY IN THE BRAIN, WHAT
ADVANTAGES, WE'LL SAY THIS, IN
NEUROBIOLOGY, NEUROBIOLOGISTS WE
MAKE IN OUR BRAINS WE MAKE MANY
MORE CELLS DURING DEVELOPMENT
THAN WE ACTUALLY NEED.
SO THERE'S A SUPERNUMERATION
THAT OCCURS IN THE PERIPHERY AND
THE CNS AND THEN THERE'S A DIE
BACK THAT OCCURS OF THESE CELLS.
SO YOU ACTUALLY MAKE MORE
NEURONS THAN YOU -- THE MORE
CELLS ARE PROLIFERATING.
SO ONE ARGUMENT THAT'S MADE
ABOUT SUPERRER NUMERATION WHICH
IS A SEPARATE PHENOMENA IS THE
BRAIN ACTS ON THAT SUPER
NUMERATION TO SELECT FOR THOSE
CELLS THAT HAVE SOME ADVANTAGE
OR ARE BETTER WITHIN THE POOL.
AND WHAT THIS MAY TELL YOU IS
THAT DOESN'T TELL YOU WHAT
THEY'RE SELECTING ON.
WHAT IS THE VARIANCE ON WHICH
THE TARGET MAYBE SELECTED THOSE
CELLS GENERATED.
THIS MAYBE ONE OF THE MECHANISMS
THAT PROVIDES SOME DIVERSITY
WITHIN THE POOL OF PROGENITOR
POPULATIONS ON WHICH THE LOCAL
TARGET AREAS THEN COULD SELECT
FOR STOCHASTIC -- SELECT FOR
ADVANTAGE DURING THE
DEVELOPMENTAL PERIOD.
>> BUT IN THAT CASE DON'T YOU
THINK THIS WILL NOT BE A RANDOM
PROCESS OF STOCHASTIC PROCESS?
IN THAT CASE IF YOU SELECT FOR
SPECIFIC NEURONS IN SPECIFIC
PARTS OF THE BRAIN THIS, HAS TO
BE A LITTLE BIT MORE REGULATED
AND (INAUDIBLE) MAY OCCUR IN
SPECIFIC REGIONS IN DIFFERENT
TISSUES OR DIFFERENT CELL TYPES
TO GIVE THAT SELECTIVE
ADVANTAGE.
OTHERWISE IF IT'S A RANDOM
PROCESS THE SUPERIOR --
>> YOUR POINT WE HAVE THIS
DISCUSSION A LOT AND THE POINT I
THINK IS WELL TAKEN, I WOULD SAY
EVOLUTION IS NOT TARGETED.
THIS WOULD BE A WAY OF HAVING
MORE OF A BROAD RANGE OF
OPPORTUNITIES OF CHANGES
REMEMBER, IF IT'S 80 OR 300 THE
GENOME IS LARGE SO THERE'S MANY
PLACES THAT THEY CAN LAND HAVING
NO ACTIVITY AT ALL.
SO TAKE THE EXTREME CASE THAT
THEY ARE COMPLETELY STOCHASTIC
WHICH I DON'T BELIEVE THAT'S THE
CASE BECAUSE THERE'S TOO MUCH
CHROMATIN PACKING.
MORE LIKELY STOCHASTIC.
THAT WOULD BE THE MECHANISM
THROUGH WHICH YOU GET A BROADER
RANGE OF DIVERSITY ON WHICH
SELECTION COULD OCCUR.
ALTERNATIVELY, THAT'S WHY WE'RE
DOING THE DEEP SEQUENCING, IS
THERE MAY IN FACT BE HOT SPOTS,
THIS -- THERE MAYBE REGULATED
BUT I'M BEING AGNOSTIC ABOUT
THIS RIGHT NOW BECAUSE I DON'T
KNOW THE ANSWER IN FACT.
SO I'M OPEN TO BEING ABLE TO
EXPLAIN EITHER WAY BUT WE'RE
ANTICIPATING NOT HOPING BUT
ANTICIPATING THAT THERE MIGHT BE
SOME SPECIFICITY FOR INSERTION
AND THAT WOULD BE INTERESTING.
THAT'S WHAT WE'LL FIND OUT.
SO WE'RE IN EARLIER STAGE.
SORRY IT'S LESS SATISFYING AN
ANSWER THIS POINT.
>> I HAVE ONE QUESTION.
>> WE'LL HAVE A LAST QUESTION
AND THEN CONVENE THIS IN THE
LIBRARY FOR A RECEPTION.
>> I JUST HAVE ONE QUESTION
REGARDING THE SOX2 AND YOU SAID
THAT LINES SOX2 MEDIATED
TRANSCRIPTION AND THERE IS A
CHANGE IN EPIGENETIC CHANGES
ASSOCIATED WITH THE METHYLATION
AND ACETYLATION AND HOW IS THE
NEURAL DIFFERENTIATION IS
TRIGGER AND HOW IS IT THAT IN
THE SOX2 KNOCK OUT IS THERE A
CHANGE ANYTHING LIKE?
YOU SEE THE CHANGE IN THE NEURAL
IMPLEMENTATION?
>> SOX2 KNOCK OUT ARE LETHAL
VERY EARLY ON SO THERE'S A
HYPOMORPH AND WE HAVEN'T LOOKED
THAT THE BUT YOU THINK ABOUT IT
IN THE RIGHT WAY.
JUST THEY DIE SO EARLY THAT WE
HAVEN'T BEEN ABLE TO DO THAT, IF
WE KNOCK OUT SOX2 IN VITRO WE
CAN SEE THE SAME SORT OF
INCREASE IN LINE ACTIVITY BUT WE
ARE NOT ABLE TO DO THAT
EXPERIMENT IN VIVO AND WE
HAVEN'T LOOKED AT THE HYPOMORPH
TO SEE WHAT HAPPENS.
YOU THINK ABOUT IT THE SAME WAY
WE ARE.
THANKS.
>> THIGH.
>> OKAY.
SURE.
[APPLAUSE]
>> WE WILL CON CONVENE IN THE
LIBRARY.