2. MARCO TEÓRICO
2.4 LOS PRODUCTOS ORGÁNICOS EN EL ECUADOR
2.4.1 MARCO JURÍDICO
Most gene targeting experiments aim to completely ablate gene function via the introduction of a null m utation into the gene of interest. The generation of a null m utation usually involves either the introduction of a targeting cassette into the reading frame of the gene or deletion of a portion of the genomic locus including coding sequence in com bination w ith targeting vector insertion. In either case, insertion of a targeting cassette is designed to take place as close to the start of translation as possible to prevent the p ro d u ctio n of truncated proteins that m ay possess partial function and therefore obscure phenotypic analysis. H ow ever, m any genes exhibit variability at the 5' end of their transcript composition via usage of alternative transcriptional or translational start sites or alternative splicing events. Precautions have to be m ade to ensure that targeting vector insertion does not occur w ithin an alternatively spliced exon or u pstream of an alternative translational start site, since in either scenario, the m u tatio n m ay be elim inated from the read in g fram e w hich remains essentially intact.
In the case oi MU, the translational start is located w ithin exon 1, w hich by
extrapolation w ith the position of the same exon of its hu m an hom ologue, is
anticipated to lie approximately 35kb upstream of exon 2 (Gu et a l, 1992). Exons
1, la and 2 are all particularly short in length and do not encode any previously
described motifs currently thought to influence MU function. H ow ever, both
exons la and the large exon 3 have been reported to exhibit alternative splicing and therefore are not good candidate exons for targeting vector introduction
(Domer et al, 1993). Instead, the region encoding the P H D /L A P dom ain was
selected for further characterisation as a site for targeting vector recombination. Four lines of evidence suggested that rem oval of both the P H D /L A P finger
structures and the carboxy term inal SET dom ain of MU could potentially
generate a null m utation with no significant functional activity. Firstly, there is a high degree of sequence conservation of the PH D /L A P and SET dom ains betw een flies and m am m als despite significant sequence divergence in other
parts of the protein (Gu et al, 1992; Tkachuk et a l, 1992). Secondly, the MLL
BCR is com m only targeted by m ultiple chrom osom al translocations and in freq u e n tly by d eletio n s, b o th of w h ich are asso ciated w ith acute leukemogenesis. These structural alterations to the MLL gene result in either the partial loss of one of the three PH D /L A P fingers or com plete loss of both the P H D /L A P dom ain and the SET do m ain as w ell as all in terv en in g
Chapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
sequences. Thirdly, the PH D /L A P dom ain is subject to post-transcriptional regulation in the form of two types of alternative splicing involving the first
and third PH D /LA P fingers (Lochner et ah, 1996; Ma et al, 1993). Fourthly and
m ost im portantly, homozygote flies carrying the trx^^^ deletion allele resulting in a fram eshift m u tatio n im m ediately u pstream the P H D /L A P and SET
dom ains of Drosophila trx are embryonic lethal, unlike the trx^3 allele w hich
results in the in frame deletion of 278 amino-acids betw een the P H D /L A P and
SET dom ains (Mazo et al, 1990; Breen and Harte, 1991).
MLL chrom osom al translocation consistently resu lts in fu sio n p ro te in p roduction, b u t also loss of the carboxy term inus of MLL including the conserved PH D /L A P, transactivation and SET dom ains. A nalysis of MLL partner proteins initially identified had revealed few sim ilarités and it seem ed difficult to predict a com m on them e to explain their co n trib u tio n to the transform ation potential of MLL fusion proteins. Thus it rem ains equally plausible that the mechanism for oncogenic conversion of MLL could include either the production of a dom inant gain of function fusion protein, or loss of MLL function w holly via d o m in an t negative in h ib itio n or p artially via haploinsufficiency. To investigate the latter tw o possibilities, a targ etin g
strategy was undertaken to truncate the MU protein im m ediately upstream of
the equivalent BCR, so that such a truncated MU p ro tein w o u ld lack the
PH D /L A P, transactivation and SET dom ains and in some respects mimic the effect of chrom osom al translocation u p o n MLL gene stru ctu re w ith o u t the addition of partner gene sequences.
Vector design
Restriction m apping studies locating a unique BssHII site w ithin the coding
sequence of MU exon 5 (nucleotide 3584 of MU cDNA sequence), im m ediately
upstream of the MU equivalent BCR and thus identified this site as a prim e
candidate for the insertion of a selectable m arker gene. Restriction m apping studies had also positioned exon 5 w ithin a 5.5kb EcoRI restriction fragm ent, w hose length w as sufficient to provide sequence hom ology flanking exon 5 to facilitate HR. It w as also noted that this fragm ent encom passed m ost of the equivalent BCR which, in hum ans has the ability to recombine via translocation w ith a ho st of o th er p a rtn e r gene loci and m ay th erefo re rep re se n t a "recombination hotspot".
Truncation of the MU open reading frame at exon 5 w as accom plished by the
introduction of an IRES-LacZ SV40polyA-MClNeomycin targeting cassette into the BssHII site. The cassette contains the following features in 5' to 3' direction. Firstly, the 5' end of the cassette contains stop codons in all three potential
Chapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
read in g fram es so th at after insertion, prem atu re translational term ination ensues. Secondly, the 1RES sequence is placed im m ediately upstream of the |3- galactosidase rep o rter gene. The 1RES sequence allow s CAP in d ep en d en t ribosom e binding and translation of the ^-galactosidase gene to take place. A separate ^-galactosidase protein is generated w ithout concom itant fusion to am ino term inal sequences of the targeted gene. H ow ever, for p-galactosidase reporter activity to faithfully reflect the expression pattern of the targeted gene, the cassette m ust be integrated into an exon to allow p-galactosidase production
to be u n der the transcriptional control of M il Figure 4.2 illustrates the potential
effect of targeting vector integration into the BssHII site of exon 5. The plasm id linker sequences inserted during cloning betw een the BssHII site and the start of the 1RES, result in the production of 9 novel residues after w hich translation is term inated.
C onstruction of MU exon 5 targeting vector
A 5.5kb EcoRI fragm ent containing genomic DNA betw een intron 4b to intron 8 w as cloned from phage clone LI into the pUC19 vector to generate the plasm id pUC19 5.5 RI. This particular plasm id vector was selected as it does not contain
a BssHII site and therefore renders the BssHII site w ithin MU exon 5 unique. To
facilitate the introduction of the Ires-LacZ-Neomycin targeting casssette (a gift
provided by A.Smith) into the BssHII site of MU exon 5, the cassette had to be
initially m odified to generate flanking BssHII com patible restriction sites to allow cloning into a BssHII site. Firstly, a derivative of plasm id Bluescript (pBS) w as generated by the cloning of a double stranded oligonucleotide betw een Xbal and Sail sites of the poly linker to generate the plasm id pBS Oligo. The double stran d ed oligonucleotide contained the following sites from 5' to 3' respectively: Xbal-Ascl-Bglll-Ascl-Sall. The AscI site is compatible w ith BssHII. Bglll is compatible w ith BamHI and not present w ithin pBS vector.
The IRES-LacZ SV40polyA-MClNeomycin targeting cassette w as excised from pBS w ith BamHI and ligated into the com patible Bglll site of pBS oligo to generate the plasm id pBS oligo lacZ. Finally, pBS oligo lacZ insert w as cut out w ith flanking AscI sites to liberate the IRES-LacZ SV40polyA-M ClNeomycin
targeting cassette and w as ligated into the unique BssHII site of MU exon 5
w ithin pUC19 5.5 RI to yield the plasm id MU exon 5 targeting vector. Products
of each of the above cloning steps w ere restriction m apped and sequenced before continuing w ith the further cloning steps. The targeting vector w as linearised at the unique Sail site w ithin the polylinker at the 3' end of the vector prior to electroporation, resulting in the retention of pUC 19 vector backbone sequences at the 5' end of the construct.
Chapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
Figure 4.2 The predicted molecular effects of M U exon 5 targeting vector recombination upon the reading frame of the targeted M U allele.
The integration of the targeting cassette into exon 5 sequences results in the in
frame fusion of linker sequence encoding nine novel residues to the MU reading
M //exon 5 targeted genomic sequence: CAG GTC AGC GCG C / C T CGC GAA /G A T CGC GGG CCG CTC TAG
MUexon 5 targ eted am ino-acid sequence : Q V S A P R E D P G P L *
Chapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
Identification of a single copy M U probe that lies external to the 5’ end of the
M U exon 5 targeting vector
Prior to em barking upon transfection experiments, it was necessary to locate a
restriction fragm ent w ithin the MU locus which could be used as a single copy
probe for Southern blot genotyping of transfected ES cell clones. Such a restriction fragm ent should be gene specific and not contain any repetitive elements. Most im portantly, a single copy probe m ust also be located external to the targeting vector so that it is capable of discrim inating betw een random and gene specific integration.
C oding sequence does not generally contain repetitive elem ents and are therefore a rich source of single copy probes. Various restriction fragm ents derived from the 5' region of phage clone LI were screened for the presence of single copy probes to use for Southern blot genotyping. Figure 4.3 shows results of hybridising a 700bp H indlll-K pnl fragm ent term ed probe A, to a Southern blot of various restriction digests of mouse 129 strain ES cell genomic DNA. The 5' H in d lll site of probe A is located 114bp upstream of the 3' boundary of exon 3, w ith the rem aining sequence containing exon 4a, continuing to a K pnl site located in intron 4a. Probe A lies approxim ately 2kb upstream of the 5' EcoRI
site of the MU targeting vector.
Probe A was able to hybridise to a single band in each of six separate restriction digests, confirming that none of the six restriction enzym e sites w ere present w ith in the 700bp probe sequence. The size of the in d iv id u al b a n d s also corresponded exactly to that predicted by the restriction m ap generated in the
previous chapter, confirm ing the structure of MU phage clone LI coincided
w ith that of the endogenous ES cell genomic MU locus. Finally, the ability of
probe A to hybridise only to restriction fragm ents from the MU locus present
w ithin MU phage clone LI argues against the presence of either m ultiple
m urine MU homologues or MU pseudo genes.
Southern blot strategy to screen for homologous recombinant ES cell clones
A sim ple m ethod to identify hom ologous recom binant clones relies u p o n targeting vector introduction of novel restriction sites not present w ithin the germline locus of the gene of interest, which can d isrupt the size of particular restriction fragm ent length polym orphism s (RFLPs). Such alterations to the germ line p attern of RFLPs can be detected using Southern blotting. It is preferable to introduce novel restriction sites into the locus as detection of shorter m utant RFLPs can only occur after HR has taken place. If a screening
Chapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
Figure 4.3 Identification of a single copy probe that lies 5' and external to the
MU exon 5 targeting vector.
M ouse strain 129 genomic DNA w as digested w ith a ran g e of restriction enzym es, separated on a 0.7% agarose gel. Southern blotted and hybridised to external probe A. Lanes 1-5 correspond to genomic DNA digested w ith EcoRI, EcoRV, HindUI, Kpnl, PstI and S ad respectively.
- 17.7kb
- 13-Okb
- S.Okb
- 4.5kb
- 1.8kb
1
2
3
4
5
6
Chapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
strategy relies upon the deletion of a restriction site already present w ithin the germline of the locus, problem s m ay be encountered in distinguishing betw een a true recom binant clone and a random ly integrated clone w hose DNA has only been partially restricted. The strategy used to screen for recom binant ES
cell clones of the MU locus is schematically depicted in Figure 4.4. Only those
restriction sites relevant to the screening proceedure are included.
G418 titration
Prior to the in troduction of a targeting vector into ES cells, the optim al concentration of G418 required to facilitate positive selection of transfected cells had to be calculated. The optim al m inim um dose of G418 required to kill 100% of non transfected ES cells was determ ined by subjecting no n transfected p aren tal ES cells to various doses of G418 selection, ranging from 0.05 to Im g /m l in 0.05m g/m l steps. These cells were cultured u n d er norm al ES cell grow th conditions upon a STO fibroblast feeder layer u n d er selection for 10 days, after which the num ber of surviving ES cell colonies w ere determ ined. It was found that at a concentration of 400pg/m l G418 selection and above, no ES cell colonies su rv iv ed and therefore all su b seq u en t ES cell transfection experiments utilised positive selection of transfectants w ith 400pg/m l G418.
Conditions for electroporation
Conditions for electroporation of targeting vectors into ES cells were optim ised from previous laboratory protocol. Initial attem pts to electroporate ES cells
w ere perform ed using the protocol of P hilpott et al, (1992), alth o u g h the
efficiency of electroporation was found to be relatively poor, as assessed by the n u m ber of ES cell colonies present after 10 days of positive selection. On average, betw een 30-60 G418 resistant ES cell colonies w ere derived from transfections utilising such conditions. Since the estim ated average frequency of hom ologous recom bination for replacem ent targeting vectors is so low, high transfection efficiencies are particularly im portant to the success of a gene targeting project. M odifications of the original protocol w ere m ade to try to im prove transfection efficiencies. Variation of the tem perature of incubation of ES cells before and after electroporation w as found to be critical to higher transfection efficiencies. Incubation of ES cells at room tem perature before and after electroporation instead of on ice led to a corresponding > 10 fold increase in the num bers of G418 resistant colonies after positive selection. Increased transfection efficiency enabled screening of larger num bers of ES cell clones for the identification of rare hom ologous recom bination events. Increased transfection efficiency also allow ed the selection of those ES cell clones
Chapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
Figure 4.4 S outhern b lo t strategy to id en tify hom ologous reco m b in an t MU
m utant ES cell clones.
(a) Genomic organisation of the MU locus. Only those restriction sites relevant
to the screening proceedure are shown.
(b) Structure of the MU exon 5 targeting vector.
(c) Predicted stru ctu re of the m u ta n t M U allele after d o u b le reciprocal
Germline configuration of the MU locus I k b
1 I
w œ £ E X uj ceI//I
A
l
l | i
1 I
ëI i
X X m w wH
E i 8.5kb 17.7kb H in d lII —► Bam H l ► E coR V Bam HI 6 k b -EcoRI 8kb M U ex o n 5 T a rg e tin g V ecto r p U C 1 9 1RES LacZ pA N E C111
T a rg e te d A llele 4a 4bI 1
Probe A 3.5kb 1RES LacZ pA N EC Probe B ► H in d lII H Bam HI 11 9-11 P rob e C EcoRV EcoRI 6kbChapter 4 Generation of an MU mutant mouse strain P.M. Ayton 1998
possessing the m ost undifferentiated m orphology for screening and therefore im proving the chance of hom ologous recom binant ES cell clones m aintaining their totipotency, resulting in germline contribution.
Introduction of the MU exon 5 replacem ent targeting vector into ES cells and
su b seq u en t screening for recom binant clones
Once conditions for the electroporation of ES cells had been m odified to allow ad eq u ate transfection efficiency, a n u m ber of ES cell transfections w ere