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133 Homologous Recombination (HR)

1. Modification of the pw25 plasmid from Gong and Golic, 2003.

Vector PHSS7, containing two LoxP sites in the same orientation, was used for the subcloning (PH7-loxP).

The product of the annealing between the primers BamHI-AscI-Stop

(GATCCGGCGCGCCCTAGACTAGTCTAGC) and rc-BamHI-AscI-Stop

(GATCGCTAGACTAGTCTAGGGCGCGCCG) was cloned in the PHloxP vector (digested with BamHI) to produce the plasmid PHB6.

The product of the annealing between the primers EI-SpHI-Stop

(AATTCCTAGACTAGTCTAGGGTACCGCATGCC) and rc-EI-SpHI-Stop

(AATTGGCATGCGGTACCCTAGACTAGTCTAGG) was cloned into PHB6 (digested EcoRI) to produce the plasmid PHB6/ES8.

An EcoRI fragment containing the attP was subcloned from pTAattP (Groth et al., 2004) in the plasmid PHB6/ES8 to produce the plasmid PHBE-attP.

The yellow SalI fragment (5165pb) was isolated from pSUPorP (provided by PK Geyer, (Roseman et al., 1995)) and subcloned into the pBluescript vector (digested with SalI) to give the plasmid Ks(-)Yellow.

The XhoI-ClaI yellow fragment was isolated from Ks(-)Yellow and was introduced into the PHBE-attP vector to end up with the PH-Y plasmid.

An AscI-NotI fragment from PH-Y (containing the yellow reporter gene flanked by the two loxP, and the attP integration site) was used to replace the AscI-NotI fragment containing the white reporter gene in the pw25 to create the py25 plasmid.

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Map of the two deletions we initially plan to generate by HR with the py25 plasmid:

2. Homology regions:

We originally planned to create two deletions by homologous recombination, one deleting 19.3kb (iab-6 deletion), and a smaller one deleting 7280bp (Fab-6 deletion). To this end, we created two donor plasmids, Py-del Fab-6 and Py-del iab6, containing the same 4297bp homology sequence to the iab-5 domain adjacent to the proximal breakpoint of these mutations. This fragment was cloned in next the attP site (see figures below). The homology regions for the distal side of the two deletions, were clones next to a LoxP site, placing the yellow reporter gene in between the two homology regions. Py-del iab-6 contains a 4436 bp Fab-7 homologous region, and Py-del Fab-6 a 4440 bp iab-6 homologous region. The homologous sequences were generated by PCR using the primers below.

The PCR reactions were made with genomic DNA (isolated with the DNeasy blood and tissue kit, Qiagen) from the same yw flies used for the injection.

Primers sequences:

IAB7-AvrII: CCTAGGCGGCGAACAGTAGGGAAG Fab7-AscI: CAGCAAAAATCGTAAAAAAG

IAB5-NotI: GCGGCCGCGGTCAGTAAACGGGTCCC IAB5-SpHI: GCATGCACTGGCGACATTTCTC

IAB6 AscI: GGCGCGCCCGGAAGGGCTATCTTGGC IAB6 AvrII: CCTAGGCTCCGGGAAAATGGCTAC

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The two plasmids Py-del Fab-6 and Py-del iab-6 were each injected together with the turbo transposase plasmid into yw embryos.

Resulting Py+ donor lines used for the HR:

From the injection of the Py+ donor (Py-del iab6), I have used two fly strains with one donor inserted on Chromosome II (line D, color coded in orange), and the other with the donor inserted on the X Chromosome (line C, color code purple)

From the injection of the Py+ donor (Py-del Fab6), I obtained two fly strains with the Py+

donor. One is inserted on Chromosome II (line 2, color code yellow), and the other inserted on the X Chromosome (line 1, color code green)

The following illustration presents the two recombination events that should occur between the homology region carrying by the Py+ donor and the targeted 3R chromosome arm, in order to get an iab-6 deletion.

136 3. Fly work: crossing procedure for the HR

The ends-out homologous recombination (HR) was performed as described in the paper of Gong and Golic in 2003. The potential correct homologous recombination events are easily recognized. They exhibit yellow+ pigmentation in the A5 and A6 segment, while the other segments of the fly are yellow. I have recovered 15 candidates from the crosses with the orange donor, seven flies from the crosses with the purple donor, 7 flies from the crosses with the yellow donor, and only one fly from the crosses with the green donor.

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I initially planed to identify HR events by doing PCR reactions on single fly DNA extract for each of the candidates, by using specific primers for sequences outside of the homologous regions. Using this technique, I identified two HR events (3 and 25 from orange cross, see table below). I obtained perfect PCR products (which were sequenced) for each of them, but the 2 candidates were exhibiting dramatically different phenotypes. Then I decided to check if the iab-6 sequences were still present in each of the two candidates. By doing PCR with specific primers for the iab-6 sequence on homozygous flies for the two candidates, I detected specific PCR amplification products for the sequence that should be deleted if the HR occured correctly. Clearly the two candidates isolated, as well as the other, did not contain a correct HR event as expected.

Because the expression of the yellow reporter gene is only visible in the A5 and A6 abdominal segments means that at least the reporter construct is inserted in the BX-C, and more precisely, in a regulatory region silenced in A4 and more interiorly. We believe that, in each isolated line, a recombination event occurred with one of the two homology regions of the Py+ donor.

I still do not have any explanation for the recovery of so many candidates with so many phenotypes. I learned recently that this occurs frequently with the ends-out technique. I also do not really understand why I got the expected PCR products but as I did not sequence the entire iab-6 region to understand this, I can only speculate that some sort of rearrangement might have occurred within the recombined region, creating an interior duplication.

138 List of the candidates:

To try to better characterize all the candidates, we performed genomic southern analysis.

139 Genomic Southern Blot:

Analysis of candidates obtained after homologous recombination.

For this analysis we used the Rediprime II Random Primer Labeling System, the redivue (alpha32P)dCTP, the illustraTM ProbeQuant G-50 Micro Columns, Radiolabeled Probe Purification Kit and the Rapid-hyb buffer from GE Healthcare (or Amersham). Using these tools we were able to discover recombination events that were correct on one side of the deletion or the other. By isolating recombination events that occurred correctly on each side independently, we were able to recover the correct iab-6 deletion by using the loxP sites in each recombinant (see schema bellow).

First, we carefully identified molecularly the recombination events of the original lines. Using an Fab-7 DNA probe, we identified only one recombination event that correctly takes place on the Fab-7 side of the deletion. This unique candidate (6 orange) has no visible phenotype and flies homozygous for this mutation look wild type. Using the EJ probe (see below), we identified several recombination events on the iab-5 side that could have been used for the next step. The 9 orange, 9 green, and 7 yellow lines correspond to flies that do

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not have a strong iab-6 phenotype and were used in the subsequent steps (contrary to 25 and 27 orange).

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For most of the candidates, the recombinant bands were found together with the wild type bands. This suggests that in most of the cases, the iab-6 sequence is still present, in addition to the enigmatic events of recombination. Other southerns confirmed our presumption, that iab-6 sequences are still present in each of the candidates.

Recombination via the Cre Recombinase

For these experiments (described above), we start with flies in which the yellow reporter gene has been removed. These lines, therefore, contain only a single loxP site on each chromosome. The “floxed” flies lines were named 6A-B-or C. I established between 30-40 individual crosses between 6-orange ♀ or ♂and either 9-orange, 9-green or 7-yellow ♀ or

♂. The Cre recombinase was provided by either the male or the female (carried on second chromosome).

As the all starting lines displayed no or little noticeable phenotype and the resulting line should be iab-6, we first screened for the iab-6 phenotype. All candidates were first screened by PCR (with the primer pairs iab-5RH/Fab-7X2 and initiator5/iab-7RH) and then by genomic southern blot. With the genomic southern blot, a lot of candidates looked fine (all candidates were noted A, B…Z). But with the PCR reaction only 2 candidates N2 and Q recombinants seem to be perfect deletion events. The N2 and Q recombinants were obtained from two different 6B X 7C crosses. We sequenced the breakpoints of the two recombinants, to be sure that the attP insertion site was intact. Based on the phenotype of the N2 and Q flies, the mutation was named iab-5,6CI (see result section, chapter IV)

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Integration:

Cloning for re-insertion in the iab5-6CI flies

The attB sequence, plus one loxP was cloned from the attB(FL)loxP (provided by Fabienne Cléard). The fragment was excised as an Acc65I-SacI fragment where the Acc65I site was blunt ended with Klenow prior to SacI digestion. This approx. 400bp fragment was then cloned into the pKs-Y plasmid, previously cut with EcoRV and SacI. The resulting plasmid was called Ks-Y attB-LoxP.

~500bp PCR products were cloned from each of the iab-5 and iab-6 regions (starting from the breakpoints of the iab-5,6CI deletion) and linked by overlap PCR through sequences shared in the internal primers. The result of this overlap PCR is an ~1 kb fragment with homology to both iab-5 and iab-6, flanked with NotI sites and with an internal PmeI site.

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These homology sites can then be used in recombineering experiments to capture the region deleted in the iab-5,6CI deletion (see scheme below). The primers are used for the PCR reactions are listed below.

Primers used:

Iab-5 N: ATAAGAATGCGGCCGCGGTGCGTTTCCATTTTCCCTAGG

Iab-5 new (+PmeI) :CTCACCATAGAGCACCACGTTTAAACGTCGTCCGGAAATGGCAACCAG Iab-6: CTTTGCCAGCTTTTGCCACTCGTCC

Iab-6 P (+PmeI): GGTTGCCATTTCCGGACGACGTTTAAACGGTGAAGGCGCGAAACTGTGAC

The PCR products were cloned in Ks-Y attB-LoxP. The PmeI site was used to linearize this vector for the recombineering procedure. The sequence to be captured was from the Abd-B region-containing BAC, BACR24L1 (BACR24L18, GenBanK: AC095018), using the procedures described in (Lee et al., 2001; Liu et al., 2003).

The plasmid containing the 19.3kb region of iab-6 was named KsY-iab6H. The result of the integration of the 19.3kb sequence in the iab5-6CI embryos, is a perfect rescue of the deletion phenotype (iab-6rescue flies).

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1. Generation of a series of deletions in the 19.3kb sequence

The KsY-iab6H was modified using the recombineering. We generated PCR fragments containing a Kanamycin selector gene surrounded by two FRT recombination sites using primers containing 5’ leaders of 50bp that are homologous to the region flanking the sequences we want to delete. The FRT-kanamycin-FRT DNA template for PCR is provided by the pGEM1-K7-FRT-Kan-FRT (provided by François Spitz). Upon recombineering on the KsI-iab6H plasmid, the plasmids carrying the designed deletion can be selected on Kan plates.

After selection of deletions the region of recombineering is sequenced to verify correct events. The Kanamycin cassette is then removed using a bacterial strain expressing the flipase enzyme under an inducible arabinose promoter (EL 250) (Lee et al., 2001). Cleaned DNA preps are then made and used as material to inject into the iab-5,6CI flies stocks, containing an X chromosome expressing φC31 integrase enzyme under the control of the vasa promoter. (http://www.frontiers-in-genetics.org/flyc31/).

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The ΦC31 integrase (derived from a Streptomyces phage) mediates unidirectional site-specific recombination between its attB and attP recognition sites. The integration rearranges the attP site into attL and attR sites flanking the integrated sequence (Thorpe and Smith, 1998).

Oligos used for the targeted deletion:

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Position of the homology region contained in the different oligos relative to the iab-6 19.3kb sequence:

Deletion that have been generated:

All the deletions, generating by using the FRT-Kan-FRT cassette, contain a few base pairs of DNA sequence necessary for the annealing of the homology regions. That is why, the iab-5∆1 deletion contained 158bp more on its proximal side than the iab-5,6CI deletion, and the iab-6∆5 deletion contained 173bp more on its distal side than the iab-5,6CI deletion. This does not mean that any sequence is duplicated, just that we cannot remove sequence at the very edge of the iab-5,6CI deletion.

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Identification of putative binding sites at the iab-6 initiator locus.

We used the Ahab algorithms to detect cis-regulatory modules. This program scans the 19.3 kb of iab-6 sequence for clusters of transcription factor binding sites (http://gaspard.bio.nyu.edu/fly_ahab.html). Ahab detects three cis-regulatory modules in the 19.3kb sequence, and one of them (module Ahab_12391) was localized in the 2.8kb OT sequence (or 2.8XN) previously identified in transgenic assays as the iab-6 initiator (Mihaly et al., 2006). This module is deleted in the iab-64 deletion.

We decided to create three new mutant flies each of them containing the 19.3kb sequence but mutated for the Caudal, Krüppel or Hunchback putative binding sequences in the initiator. We generated three mutated plasmids: one mutated for all three putative Caudal binding sites, another mutated for all four putative Krüppel binding sites, and one delete for 24bps containing the two putative hunchback binding sites.

For the mutagenesis, we designed a plasmid containing the initiator sequence flanked by 500bp of surrounding homology sequence, plus a kanamycin selection cassette (plasmid amazingly named “KS+short target for mutagenesis(-)KanFRTblunt”). We used the QuikChange Multi Site-Directed Mutagenesis Kit from Stratagene.

150 Strategy:

151 Sequence of the primers used for the mutagenesis.

The figure below presents for each binding site mutated: the wild type sequence of the initiator with the putative binding site (in capital letter), the base pairs mutated in blue. Below the wild type sequence, the mutated sequence of the oligos with the restriction site created in red, and deleted base pairs represented by a horizontal dash.

152 In silico data

1. Results of the analysis using the Fly_Ahab webserver:

The following table has 8 TAB delimited fields. As an example, I will describe the first line.

The first field "Ahab_7171" denotes that Ahab predicts a cis-regulatory module centered at position 7171 in the sequence (in this case, the 19,3kb iab-6 sequence). This position is relative to the local coordinates of the analyzed locus, in this particular case, it is the 19.3kb iab-6 sequence. The score of this module is 22.985614 (the last entry of the last field). The second field tells us that we are looking at a binding site prediction for the transcription factor knirps. This binding site is located at position 12712497-12712511 in the melanogaster genome (4th field). The probability that this site is bound by knirps is 0.8 (4th field), and it resides on the - strand (5th field). The last field reiterates the name of the transcription factor and provides the actual sequence of the binding site and the overall score for the predicted module of which this binding site is part.

Because the Ahab webserver (http://gaspard.bio.nyu.edu/fly_ahab.html) is not accessible anymore, we are not sure that the list of the binding sites used by the Fly_Ahab webserver is exhaustive. We believe that Fly_Ahab looks for bicoid, caudal, dorsal, hunchback, knirps, kruppel, tailless, fushi tarazu (ftz), even skipped (eve-en), Stat92E (Dstat), paired (paired-HD), torRE (Torso-response element), giant and odd skipped.

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Position of the putative binding sites found by the Ahab algorithm, on the 927bp sequence deleted in the iab-64 mutant:

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We also looked at the ChIP on chip data provided on the 19.3kb iab-6 sequence through the UCSC genome browser. Those results are shown below.

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