All care and m aintenance of mouse stocks were perform ed by staff at the ICRF Transgenic U nit, Clare H all Laboratories in accordance w ith H om e Office Regulations. Young mice w ere w eaned at three w eeks of age. M ouse tail biopsies were also harvested at weaning.
PGR genotyping of M U and p53 mutant mice
Mice were genotyped following a modified version of the protocol described in chapter 2. Routinely a IX PGR buffer mix was prepared as described in chapter 2. For each reaction, the IX PGR mix was supplem ented w ith 0.2|xl of 5U/jLil Taq
polym erase and lOOng of oligonucleotides 5D, 5E NEC F8 for MU and 5N, 3W2
and NEO F8 for p53 m utant alleles, whose 5' to 3' sequences are listed below:
Oligonucleotide NEO F8 : T G I GGT G IG ATG TGA GGT TGG
Oligonucleotide MU 5D : G IG GTT G IG GGG G IG GAG G
Oligonucleotide MU 5E : GAG GAA AGA GAG GAG T IG T G I G
Oligonucleotide p53 5N : G IG GGA GGG AGA AAA GTT GGA GGG G
Oligonucleotide p53 3W2 : ATG GGA GGG TGG GAG TGG TAA GGG
A complete PGR mix was vortexed, briefly centrifuged and then aliquoted into the appropriate num ber of tubes. Finally, Ip l of each genomic DNA sam ple, p repared from m ouse tail biopsies was added to each tube and mixed. Great care w as taken to avoid potential contam ination betw een sam ples. Aerosol resistant tips were used throughout the procedure and the lid of each tube was closed p rio r to the addition of the next sam ple to the adjacent PGR tube. G onditions for the PGR consisted of a 4 m inute d én atu ratio n step at 94°G,
follow ed by 33 cycles of 94°G for 20s, 62°C annealing for 30s an d a 72°G
extension step for 1 m inute w ith a final extension of 3 minutes.
Immunofluorescence analysis by flow cytometry
Gell suspensions from thym us and spleen w ere prepared by dissaggregation betw een frosted slides in FAGS buffer. Bone m arrow cells w ere harvested by passage of FAGS buffer through the bone cavity u n til the contents w ere released. Bone m arrow tissue was then sucked in and out of a 25 gauge needle
MU haploinsufficiency and haematopoietic development P.M. Ayton
and syringe to com pletely dissaggregate the tissue into single cells. Cell suspensions w ere stained for the detection of surface antigens an d then analysed by flow cytometry. FACS buffer consisted of : PBS A / 5% PC S/ 0.1% sodium azide. For the m ajority of FACS analysis, 10^ cells w ere stained per sample. Antibodies (Pharm ingen or Caltag) were generally diluted 1 in 100 in FACS buffer. A ntibodies directly conjugated to fluorescein isothiocyanate (FITC) or phycoerythrin (PE) were used at a final concentration of 5jig/m l. A lternatively, antibodies directly conjugated to biotin w ere used followed by second layer of 0.02|ig/m l strep ta vidin-T ricolour (Caltag). These fluorochromes are excited by a 488nm laser, w ith emission w avelengths of 525,575 and 667nm respectively.
All FACS incubations were perform ed at 4°C. For staining, the appropriate num ber of cells w ere aliquoted into individual FACS tubes and pelleted by centrifugation at lOOOrpm for 5 minutes. The pellet w as resuspended in lOOpl of FACS buffer together w ith a 1:100 dilution of the relevant antibody and incubated for 30 m inutes on ice in the dark. Cells w ere then w ashed free of u n b o u n d antibody by the addition of 2mls of FACS buffer, centrifuged at lOOOrpm for 5 m inutes and finally resuspended in 300pl of FACS buffer. If biotinylated prim ary antibodies were used, cells from the prim ary stain were w ashed as above but resupended in lOOpl of FACS buffer together w ith a 1 in 100 dilution of Streptavidin-Tricolour, incubated for a further 30 m inutes on ice in the dark and then finally w ashed and resuspended in 300pl of FACS buffer. A cquisition and analysis of stained cells by was perform ed on a FACScan (Becton Dickinson), using Cellquest software. For routine staining, live cells w ere gated on forw ard and side scatter on a linear scale, and data for other param eters w as collected in logarhythm ic scale. U nstained cells and cells stained w ith a single fluorochrom e were used to set up the electronic gating collection param eters. In general, 1-5 x 10^ events were collected per sample. All events w ere saved, afterw hich gates w ere introduced to allow analysis of specific haematopoietic cell populations.
Cell purification by FACS and cell cycle analysis in vivo
The proliferating com partm ent of pro-B cells w ere isolated from ad u lt bone m arrow by FACS sorting using a Becton Dickinson Vantage. Bone m arrow
m ononuclear cells w ere stained w ith the follow ing antibodies: CD43^^^c^
B220^^°^^ and HSA^^. An initial forward versus side scatter gate was included to separate lym phocytes from other bone m arrow cell types. Lym phocytes positively stained for B220 and CD43 were gated to exclude pre-B and m ature B cell populations. B220, CD43, HSA triple positive pro-B cells, corresponding to
MU haploinsufficiency and haematopoietic development P.M. Ayton
the proliferating B cell fractions B and C of B cell developm ent as defined by
H ardy et al., (1991), were analysed for cell cycle status. These sorted cells were
stained w ith Hoechst 33342 for 30 m inutes at 37°C and then analysed by FACS w ith excitation at a UV w avelength of 351-363nm, w ith H oechst 33342 fluorescence collected at 390-480nm wavelength.
MU haploinsufficiency and haematopoietic development P.M. Ayton
Results
D evelopm ent of a PCR based assay for genotyping MU m u tan t mice
Once germline transmission of the m utant allele was achieved, a m utant
MU m ouse colony w as generated. Each breeding pair consisted of two
anim als, so th at litters from such crossses p ro d u ced M //+/+, M //+ /" an d hom ozygote (M //"/') anim als in a predicted M endelian ratio of 1:2:1, the genotype of which had to be determ ined. Southern blotting is a particularly tim e consum ing and expensive m ethod for genotyping large nu m b ers of m utant mice. PCR genotyping offers the advantages of being a rapid and cost effective alternative assay to Southern blotting.
A PCR genotyping assay w as developed w hich utilised 3 oligonucleotides w ithin a single reaction to amplify two products representing both w ildtype
and recom binant alleles of M i l It is im p o rtan t to stress th a t all th ree
oligonucleotides used for PCR genotyping are located w ithin the targeting vector itself and are therefore unable to discrim inate betw een a random or hom ologous targeting vector integration. To prevent the possibility of false positive PCR genotyping of a potential random ly inherited targeting vector integration as well as that for the correctly targeted allele, mice used to set up b reed in g pairs w ere initially genotyped by Southern b lo ttin g to p ro v id e
u nam biguous genotype results concerning the inheritance of a m u tan t MU
allelle.
The PCR strategy for genotyping MU m utant mice is depicted in Figure 5.1. The
oligonucleotide set of 5E and 5D allow the amplification of a 335bp p ro d u ct
across the BssHII site of MU exon 5 and represent the germline configuration of
theM //+/+ allele. U pon integration of the targeting vector, a short extension period of 1 m inute w ithin the PCR prevents am plification of a m uch longer product from 5E, across the 5kb of the Ires-LacZ-Neo cassette to the location of the 5D oligonucleotide. However, the inclusion of a third oligonucleotide in the PCR that is specific for the 3' region of the Neomycin gene of the Ires-LacZ-Neo cassette, also allows the amplification of a 653bp p roduct from the position of
Neo F8 to 5D but only in the presence of the allele. Thus, the M//"*"/"’"
genotype will produce a single germline product of 335bp, the M il'll' genotype
will amplify two products of 335bp and 653bp corresponding to both germ line
and recom binant products and a MU~ I ~ genotype will only am plify a single
MU haploinsufficiency and haematopoietic development P.M. Ayton
Figure 5.1 D evelopm ent of a PCR based assay to genotype MU m u tan t mice
The strategy adopted to genotype MU m utants is illustrated. The position of
oligonucleotides used for PCR are depicted as arrow heads above exon 5. Exons are filled boxes. Intronic sequence is represented by thin line. The size of exons are not draw n to scale.
(a) Schem atic rep resen tatio n of PCR am plification of a 335bp p ro d u c t
corresponding to the MU germline exon 5 allele. BS = BssHII site located w ithin
exon 5.
(b) Schem atic rep resen tatio n of PCR am plification of a 653bp p ro d u c t
corresponding to the MU recombinant exon 5 allele, w ith the targeting cassette
inserted into BssHII site located w ithin exon 5.
(c) PCR genotyping of a litter of 11 anim als from intercrossing M //+/" mice. Products were resolved on a 2% agarose gel. W and H represent M //+ /+ and
control genomic DNA tem plates respectively. N ote the absence of
BS 5E E » î n 4 ^ 5D 335bp germ line pro d u ct 5E Neo FB 1RES LacZpA NEO
Exon ; 3 4a 4b 4c 5 6 - 8 5D 653bp re c o m b in æ t p ro d u ct
- 653bp
- 335bp
M 1 2 3 4 5 6 7 8 9 10 11 W HM U haploinsufficiency and haem atopoietic developm en t P .M . A y to n
M U homozygosity results in embryonic lethality
Tail biopsies were taken from a total of 25 litters containing 230 three week old F2 anim als derived from intercrossing M //+ /“ m utants. The average litter size w as 9.2. Genomic DNA was prepared from proteinase K digested tail sam ples and used as a tem plate for PCR genotyping. G enotyping results detected the generation of 61 M/Z+/+ and 167 M/Z+/" mice (genotypes for 2 offspring were not determ ined), resulting in a ratio of MZZ+/+ : M //+/" : M //"/" genotypes being 1 : 2.7: 0. Genotyping results did not identify any viable adult M //" /' m u tan t anim als, alth o u g h slightly increased nu m b ers of M //+ /" o ffspring w ere detected. PCR genotyping of a typical litter of a d u lt mice d eriv ed from intercrossing of M //+/" mice is depicted in panel c of Figure 5.1.
To investigate w hether the M/Z'/" m utation caused lethality at birth, litters of new born animals were also genotyped. N ew born litters w ere checked for the presence of any ru n ted , sick or still bo rn anim als, alth o u g h none w ere observed. G enotyping of 6 litters of new born F2 anim als derived from MZZ+/" intercrosses also failed to detect any MZZ"/" m utants. These results suggested that hom ozygote m utation of MZZ resulted in embryonic lethality.
Leukaemic chrom osomal translocations involving the MLL gene result in the loss of central and carboxy term inal portions of the MLL p ro tein and their replacement by a variety of partner protein sequences. Thus, a 50% reduction in the dosage of full length w ildtype MZZ protein m ay significantly enhance the ability of MLL oncogenic fusion proteins to subvert norm al haem atopoietic proliferation a n d /o r differentiation. Further phenotypic analysis regarding the effect of MZZ haploinsufficiency u pon adult definitive bone m arrow derived haem atopoietic development are described below. These experim ents utilised a w ide variety of monoclonal antibodies that recognise surface antigens specific for the identification of haematopoietic cell types via flow cytometry.
M U haploinsufficiency and thymocyte development
Various stages of thymocyte developm ent have been defined by the ordered rearrangem ent and subsequent expression of the T cell receptor (TCR) genes, as well as the expression of a num ber of cell surface antigens w hich can be conveniently analysed by use of flow cytom etry of viable p rim ary cells. In particular, expression of the co-receptors CD4 and CDS surface antigens have been m ost useful in devising a general scheme for T cell developm ent th at includes the progressive developm ental restriction of an im m ature double negative CD4" CDS" (DN) population initially to double positive CD4+ CDS+
MU haploinsufficiency and haematopoietic development P.M. Ayton
(DP) and then m ature single positive CD4+ and CD8+ (SP) subsets (reviewed Kisielow and von Boehmer, 1995).
The earliest thym ocyte subsets are contained w ithin the DN com partm ent, com prising only 2-3% of all adult thymocytes. DN thym ocytes undergo initial proliferation, followed by TCR y, Ô or P chain rearrangem ent. Expression of a functional in frame TCRp chain after successful rearrangem ent of one allele, results in the suppression of further rearrangem ent of the second TCRp allele, a
process term ed allelic exclusion (Uem atsu et aL, 1988). F u rth er ro u n d s of
proliferation then take place in association w ith differentiation events, leading to the p ro d u ctio n of DP com partm ent, w hich com prises the m ajority (approximately 85%) of total thymocytes.
U pon reaching the DP stage of T cell developm ent, thym ocytes u ndergo a second ro u n d of gene rearran g em en t involving the TCRa locus. A fter functional rearrangem ent of the TCRa chain, a TCR com prised of an a P heterodim er in association w ith a CD3 complex is assembled and deposited on the cell surface. The subsequent fate of the DP thymocyte is d ependent upon selection mechanisms which assess the functionality of its TCR in recognition of antigenic peptide presented in the context of major histocom patibilty complex (MHC), w hich results in either positive selection and cell survival or negative
selection and death (Bevan, 1977; Zinkernagel et aL, 1978). The ability of a TCR
complex on the surface of a DP T cell to recognise a peptide presented by MHC Class I and II molecules expressed on antigen presenting cells (APCs) w ith interm ediate affinity/avidity will result in its positive selection and survival. How ever, the ability of the majority of TCRs to recognise peptide presented in the context of MHC w ith very low affin ity /av id ity leads to their d eath by neglect (Surh and Sprent 1994). TCRs that recognise MHC presented peptide w ith high affin ity /av id ity could generate autoim m une reaction against self antigens and are therefore actively elim inated. After p rogression th ro u g h positive selection events, a DP thymocyte will dow nregulate either CD4 or CD8 to develop into m ature CD8 SP cytotoxic T cell or m ature CD4 SP helper T cell lineages. Both subsets of m ature SP thymocytes exit the thym us to populate the peripheral lym phoid organs and blood w hereupon they are able to participate in im m une surveillence and response.
Experiments were perform ed to investigate w hether MU haploinsufficiency had
comprom ised norm al thymocyte development. Thymocytes from a w hole litter of two and a half w eek old mice derived from intercrossing M //+/" mice were harvested, stained w ith antibodies to CD4 and CD8 and analysed by FACS for CD4 and CD8 surface expression. The percentage and total cell num ber of each
MU haploinsufficiency and haematopoietic development P.M. Ayton
thym ocyte subset derived from the FACS data are sum m arised in Table 5.1. This litter contained two M //+/+ and six M //+/" mice. As show n in Figure 5.2.a, M/Z+/+ thymocytes from mouse 5 displayed typical percentages of DN, DP and CD4 and CDS SP populations, accounting for 7.95, 83.96, 5.29 and 2.80% of total thymocytes respectively. Analysis of four out of six M //+/" thymocytes revealed identical percentages and cell num bers of all T cell subsets seen in M //+/+
contols, suggesting that in the majority of cases, MU haploinsufficiency did not
d istu rb thym ocyte developm ent. H ow ever, the rem ain in g tw o
thym ocyte sam ples did exhibit variable alterations to specific T cell subsets. Figure 5.2.c and d show that three of the four m ajor T cell subsets do not co n tain cell n u m b ers and p ro p o rtio n s associated w ith n o rm al T cell developm ent. M ouse 8 possessed the m ost severe effects u p o n thym ocyte m aturation, w ith m ouse 7 exhibiting a sim ilar b u t interm ediate phenotype upon the same T cell subsets. In comparison to either MZZ+/+ or asym ptom atic MZZ+/" thym ocytes, the proportion of DP cells from m ouse 8 w as reduced almost by half to comprise only 46% of thymocytes and was associated w ith a corresponding reduction in DP cell num ber by 56%. Furtherm ore, the loss of DP cells occurred in conjunction w ith an approxim ate three-fold increase in the proportion and doubling of the cell num ber of the DN com partm ent. These
results suggest that, in some cases, MU haploinsufficiency partially im pairs the
DN to DP transition.
In all six M //+/" thymocytes, the proportion and cell num ber of the CD4 SP subset w as identical to MZZ+/+ litterm ate controls. However, there w as a large expansion in the proportion and cell num ber of the CD8 SP subset in the same tw o M //+/" sam ples that contained defective DN to DP transition. M ouse 8 again possessed the m ost severe effect upon CD8 SP production, w ith m ouse 7 having an interm ediate phenotype. The proportion of CD8 SP thymocytes from