EMBO/ICGEB Phage Display Course

Paul N Hengen pnh at ncifcrf.gov
Fri Aug 23 23:44:03 EST 1996

     *** EMBO/ICGEB Phage Display Course November 17-26 1996 ***

This course is designed to give students an understanding of the use of
phage display in biological applications.  In particular, students will be
given hands on training in the principles of creating a phage library, in
the selection and subsequent screening and analysis of selected clones.
A downstream use of cloned recombinant antibodies isolated from either phage
antibody libraries or from hybridomas is in their intracellular expression to
inhibit the function of endogenous molecules.  The use of ectopic antibody
expression to inhibit protein function will be demonstrated.

The course will be divided into four modules:

1	Library creation
2	Selection
3	Screening and analysis
4	Intracellular expression of antibodies (demonstration)

Each module is divided into a number of different parts, and each student will
do at least one part from each module.  There will be extensive interactive
discussion and all students will be expected to give a ten minute seminar on
their work.

Students must have absoulute familiarity in the techniques of molecular
biology, especially DNA cloning and PCR.  Experience in the expression of
recombinant proteins would be an advantage.  Students who do not have
experience in molecular biology will find the course too difficult to
follow as no basic techniques will be explained.  Students may be pre or
post-doctoral, providing their experience is adequate.

Students from industry or academia may apply, although different costs
apply (see below).

Students from EMBO member countries (Austria, Belgium, Czech Republic,
Denmark, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Israel, Italy, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland,
Turkey, United Kingdom) working in EMBO member countries will be given
preference, although applications will also be considered from students
working abroad who will be returning to an EMBO member country, and from
students who are presently working in EMBO member countries whatever their

In addition, two to three places will be reserved for students from ICGEB
signatory countries (Afghanistan, Algeria, Argentina, Bangladesh, Bhutan,
Bolivia, Bosnia & Herzegovina, Brazil, Bulgaria, Chile, China, Colombia,
Congo, Costa Rica, Croatia, Cuba, Ecuador, Egypt, F.R. Yugoslavia, Georgia,
Ghana, Greece, Hungary, India, Indonesia, Iran, Iraq, Italy, Kyrgyzstan,
Kuwait, Mauritania, Mauritius, Mexico, Morocco, Nigeria, Pakistan, Panama,
Peru, Poland, Romania, Russia, Senegal, Slovenia, Spain, Sri Lanka, Sudan,
Syria, T.F.Y.R. Macedonia, Thailand, Trinidad & Tobago, Tunisia, Turkey,
Uruguay, Venezuela, Viet Nam, Zaire).

The application form should be filled in and returned by 15 September 1996.
This can also be done by email to bradbury at icgeb.trieste.it One recommendation
letter (sent by separate cover/email) in which the degree of experience in
molecular biology is spelled out should also be provided.  Successful
applicants will be informed by 30 September 1996.

The cost per student will be 500.000 Italian lira (to be paid in advance).
Industrial participants must pay in addition to this, 1500 AU (ITL
3,191,280 or US$ 1971.98) directly to the EMBO head office.
The cost includes all accomodation and meals as well as the course costs.

Two grants of 500.000 ITL will be provided to two students in particular
need.  These students will also have their registration fee waived.
Students wishing to be considered for these grants should explain their
reasons in an accompanying letter.

Outline of the course
Library creation
This module is divided in three: two practical parts and a theoretical
part.  The first is the creation of a phage antibody library, the second,
the creation of a phage displaying recombinant cDNA clones and the last a
discussion about peptide libraries.  Students will do one of the two
practical parts and follow the other.

1	A small phage antibody library will be made from RNA derived from
        a naive source (e.g. PBL).
2	The cDNA phage display module will involve the use of the pJuFu vector.
3	A talk will be given about making phage peptide libraries.

This module will involve the selection of binding phage antibodies, phage
peptides or cDNAs using a number of different techniques.  Each technique
will be explained and students will be divided into a number of different
groups to do one or more of each of the different selection methods.

1	Solid phase: antigen bound to plastic.
2	Biotinylated Ag with subsequent capture of the phage antibody
        /biotinylated antigen using magnetic avidin beads
3	Cellular (EGP2)
4	Theoretical talk on the use of alternative methods of selection

Screening and analysis
In this module students will characterise selected phage antibodies
(selected from a library, derived from hybridomas, or prepared in advance).
Two sorts of screening and analysis will be done:

1	ELISA using phage
2	ELISA using soluble scFv induced from infected bacteria

Intracellular expression of antibodies
A downstream use of cloned recombinant antibodies isolated from either
phage antibody libraries or from hybridomas is in their intracellular
expression to inhibit the function of endogenous molecules.  The rationale
for ectopically expressing antibodies within different cellular
compartments to inhibit the funcion of endogenous proteins will be
explained.  This will be followed by a demonstration of the expression of
antibodies in different cellular compartments (cytoplasm, nucleus, ER,
mitochondria, secreted) using the confocal microscope.

Student's own samples
Requests to bring samples by students will be considered by the organisers,
and should be indicated on the application form.
Such samples (if they are accepted) may take the form of hybridomas from
which students may wish to clone the light and heavy chain variable
regions.  If samples are accepted, students must bring with them poly
adenylated mRNA and 1mg of antigen for use in selection and screening.

EMBO/ICGEB Phage Display Course: Application form

To be returned to A. Bradbury by 15 September.  Tick appropriate spaces and
type or write clearly

Name, Institute and address

Telephone       Fax            email

Age             Tenured position      Post-doctoral    Ph.D. student

Nationality        (member) EMBO           ICGEB

Research experience

Title of the ten minute talk you will give

How will this course benefit your research?

What you will be able to offer other course members?

Two best publications

Would you like to bring a sample? and if so what is the sample you would
like to bring?

Please send to:

Andrew Bradbury
Area di Ricerca
Padriciano 99
Trieste 34012

Tel +39 40 398995
Fax +39 40 398991
email: bradbury at icgeb.trieste.it

* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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