DATE:

June 22, 2008

TO:

Marcel Dulabo

FROM:

Sébastien Vigneau

SUBJECT:

Progress report

Marcel, here is the report you requested about the advancement of my research project since I started
it on April 1st. I have included a general overview of the project and details on the progress made on
each objective.

Project Overview

Subject. The project aims at taking benefit of our mouse lines knocked-down for Toto in the male
germline to 1) investigate the involvement of Toto in controlling the expression of several imprinted loci
and 2) characterize the nuclear localization of imprinted genes regulated by Toto. My previous report
concluded that the most appropriate stages for the expression analysis are blastocysts and E6.5
embryos, and that nuclear localization should be monitored at the two-cells, morula, blastocyst and E6.5
stages.

Purpose. The purpose of the project is to characterize paternally imprinted genes which monoallelic
expression relies on the function of Toto in the male germline, and to test the hypothesis that the
propagation of the imprint set in the germline involves the localization of Toto target genes to specific
sub-nuclear compartments during early development.

Progress Overview

During the last three months, I decided which imprinted loci and genes to assay first both for their
expression and their nuclear localization, I set up expression assays for these genes and started to look
at the nuclear localization of the corresponding imprinted clusters using RNA- and DNA-FISH. Each of
these aspects is described more in detail below, along with how I plan to continue the project.

Objective 1: Find candidate imprinted genes for being regulated by Toto

Work Completed. Two sources of information are available to find candidate genes: a database of Toto
binding sites in the mouse genome (http://totobinding.org) deduced from the human consensus
sequence (Tutu et al., 2008), and a set of 4 articles characterizing the physical interaction of Toto with 4
distinct imprinting centers in mice - TIC, TAC, TUC and TOC (Titi et al., 2002; Tati et al., 2003; Tuti et
al., 2005 et Totu et al., 2008). As the binding to these imprinting centers is experimentally documented,
at least using in vitro binding assays, I decided to perform the first expression analysis for genes
adjacent to and regulated by them, that is TIG, TAG, TUG and TOG, respectively. For the same reason,
nuclear localization experiments will be performed first for the clusters including these genes.
Objective 2: Setting up expression assays

Work Completed. Before to start designing new assays, I made an inventory of the assays available in
the laboratory. In order to facilitate the access to the collected data, I organized them in an online
database to which the members of the laboratory can connect with a password. Real-time RT-PCR
assays are certainly the best solution to quantify the overall expression of the candidate genes on small
amount of material, . However, such assays had been developed in the laboratory for only TIG among
the four genes of interest. I hence designed and tested 3 other sets of primers for the remaining genes,
and all work fine. Similarly, allelic expression assays were available only for TIG. In order to find
polymorphisms between the B6 and CAST strains that can be used for allelic assays for TAG, TUG and
TOG, I designed for each gene a PCR with primers flanking a SNP annotated in Ensembl. The SNPs are
in an exon common to all known transcripts but in every case, which strain carry which allele has not
been reported. After successful amplification of genomic DNA using these PCR, I sent their product for
sequencing last Monday and should obtain the results by the end of the week.

In addition to the design of assays, I spent time to optimize the RNA extraction and the reverse-
transcriptase steps to allow analysis from single blastocysts. RNA nanoprep kits from Stratagene
(#12345) combined with reverse transcription using the Superscript Transcriptase III instead of the
Superscript Transcriptase II (Stratagene, #45678) allow amplification of the house-keeping gene Rbp0
from single blastocyst in 33 cycles using the Lightcycler, which is good enough to avoid the need of
linear amplification of the total RNA before the PCR.

Work Remaining. Based on the result of the sequencing, allelic expression assays have to be
designed. For now, I plan to design allele-specific PCR, but we can discuss further during the next lab-
meeting about the advantages of each strategy to assess allele-specific expression. Futhermore, crosses
between mutant male of B6 background and WT female of CAST background have to be set to collect
blastocysts and E6.5 embryos for the expression analysis.

Objective 3: Setting up RNA- and DNA-FISH

Work Completed. I ordered from chori.org the BACs RP123, RP456, RP789 and RP321, which include
the TIG, TAG, TUG and TOG candidate genes, respectively and extend over about 150 kb in the
corresponding clusters. Hence, these BACs also contain the genes PIG, PAG, PUG and POG, respectively.
BACs were successively amplified using the Large Construct Maxiprep kit from Qiagen (#76543) and
labelled by nick translation using the Vysis kit (#98765). As a first attempt, RNA-FISH was performed
on MEFs prepared according to Dumicro et al., 2007, and in each case two bright pinpoint signals could
be observed in more than 90% of nuclei. Similarly DNA-FISH was performed on the same cells and with
the same probes after different denaturation times. Denaturation for over 3 minutes seem to give better
results in each case.

However, performing FISH on embryos may lead to specific issues as compared to cultured cells, and I
got some useful informations about that from Marie Dupinceau. She gave me a protocol using
fibrinogen to mount early embryos on coverslip to preserve their tridimensional organisation during
subsequent FISH. I have not tried yet this protocol with our own probes, but it looks promising.

Work Remaining. The FISH protocol has to be adapted to work with embryos, likely following the
recommendations of Marie Dupinceau. Furthermore, image processing has to be set to allow efficient
segmentation of cells and nuclei and batch processing. In addition, though the use of probes
synthesized from BACs is well adapted for DNA-FISH, RNA-FISH should use instead probes
corresponding to single genes. Hence, I plan to subclone from the BACs the sequence corresponding to
the TIG, TAG, TUG and TOG genes. The easiest way is probably to perform gap-repair by
recombineering, and I would be happy if we can discuss about the technical issues related to this
technique during our next lab meeting.

Please, do not hesitate to ask me any complimentary documentation or raw data concerning the
project. I am also looking forward to having our next lab meeting and getting feedback from other
members of the laboratory.