2.2. Marco Teórico
2.2.21. Características Básicas de la Justicia Comunal
r3 (0 n o c_ Q . (D > > C - 3 on 1.00 - mammary tumour
0.75 -
any tumour0.50 -
0.25 -
0.00 - time in monthsF ig u re 3.8 G ra p h of tu m o u r incidence overall an d m a m m a ry tu m o u r incidence in all m ated m ice.
In ad d itio n to the d evelopm ent of tum ours, other effects of the transgene, as previo u sly d esc rib ed in the FI g en eratio n (B ouchard et al., 1989), w ere observed. S plenom egaly w as a com m on finding. This ap p e are d to m anifest as re d p u lp , w ith som e evidence of e x tra m e d u lla ry h aem opoiesis. In m ale m ice fertility w a s n o t affected so early. M ice con tin u ed to breed u p until 7 m onths, as com pared w ith 3-4 m onths in the FI generation m ice (B ouchard et al., 1989). A t p o st m o rtem exam in atio n m ale m ice w ere fo u n d to h av e e n la rg e d o r ab n o rm a l sem in al vesicles an d en la rg e d e p id id y m is. H a rd e ria n g la n d h y p e rp la sia w as often e v id en t a n d in tw o m ice lu n g m étastasés w e re fo u n d co n sisten t w ith H a rd e ria n g lan d carcinom a in the presen ce of an a p p a re n tly b en ig n tu m o u r of the H a rd erian gland.
C o m p a riso n c a n n o t be m a d e w ith th e n a tu ra l h isto ry of the tra n sg e n e n e g a tiv e litterm ates as the n u m b ers of m ice entailed in the stu d y n ecessitated large am o u n ts of sh elfsp ace in a sp ecified p a th o g e n free u n it. As a re s u lt only a sm all n u m b e r of tran sg e n e n eg ativ e m ice w ere k ep t for p artic u la r com parisons such as h aem atological v a lu e s.
C h a p te r Three______________________________________________________________The N a tu ra l H is to n / o f the M o d e l
Inbreeding the colony
B ack cro ssin g o n to a B A L B /c b a c k g ro u n d w as p e rfo rm e d fo r e ig h t su cc essiv e generations. W ith successive generations the incidence of m am m ary tu m o u rs declined (Table 3.2). A n u m b e r of factors m ay hav e influenced this observation, in p a rtic u la r genetic backg ro u n d . The fo u n d e r m ice, as in m an y transgenic m odels, w ere h y b rid FI m ice of C 57B 1/6/C 3H backg ro u n d . These are u se d for their litter size a n d b re e d in g vigour. The BALB/c back g ro u n d w as selected for its susceptibility to the effects of the m ouse m am m ary tum our virus. One h u n d re d percent of BALB/c mice cross-fostered onto M M TV -infected C 3H m ice w ill develo p m am m ary tu m o u rs. H o w e v er th e B A LB /c b ack g ro u n d m ay affect susceptibility to the genetic events re q u ired for d ev e lo p m e n t of spontanoues transgene-related tum ours.
FVB m ice h av e been crossed w ith h o m ozygous m ice from the n in th g en eratio n of the colony b u t after tw o consecutive m atings all offspring w ere transgene negative.
The tu m o u r incidence and characteristics of each generation are outlin ed in Table 3.2.
I
I
1
g gI
G eneration 1 2 3 4 5 6 7P roportion of transgene positive fem ales
3/4(75% ) 2 3 /3 6 (64%) 101/159 (64%) 207/392 (53%) 45/110 (41%) 5 /6 (31%) 7 /2 1 (33%)
A t risk females* 3 17 101 160 41 5
U n m ated fem ales 0 6 0 47 0 0 0
N u m b er of tum o u rs 2 13 22 46 3 0 0
^ g e of generation (m) 23 25 25 24 19 16 13
M edian age'of tu m o u r d evelopm ent (m) (range)
Too few for anaysis
20 (11-24) 18.5 (4-25) 15.5 (5-23) Too few for
analysis N o tu m o u rs to date N o tum ours to date N u m b er of m am m ary tum o u rs 1 6 15 23 3 0 0
M edian age of m am m ary tu m o u r develo p m en t (m) (range)
Too few for anaysis
17(11-20) 15 (10-25) 15 (12-23) Too few for
anaysis N o tu m o u rs to date N o tum ours to date D u ratio n of observation (m) 23 u p to 26 u p to 26 u p to 26 u p to 23 u p to 20 u p to 18
C h a p te r Three______________________________________________________________The N a tu ra l H is to n / o f the M o d e l
C hanges in colony w ith in breeding
W ith the increasing extent of in breeding onto the BALB/c back g ro u n d there ap p e are d to be a g ra d u a l change in the p ro p o rtio n of transgene positive offspring (from 64% to 31%), the p ro p o rtio n of m am m ary tu m o u rs d eveloped in transgene positive fem ales and age at th e ir d ev e lo p m e n t. (Table 3.2 d o cu m en ts the ch a n g e in the p ro p o rtio n of tran sg e n e positiv e fem ales w ith each g eneration, this is sh o w n grap h ically in F igure 3.8.). A nalysis of the second, th ird an d fo u rth g en eratio n sh o w ed a slig h t decline in the m e d ia n age of tu m o u r d e v e lo p m e n t (17, 15 a n d 15 m o n th s respectiv ely ). The m e d ia n age of onset of tu m o u rs in the w hole colony w as 18 m onths. The cu m u lativ e incidence of breast tum o u rs at 24 m onths w as 18%, the p ro p o rtio n of tu m o u rs th at w ere of m am m ary origin rem ained the sam e in successive generations. This com pares w ith a bac kground incidence of 3-5% in retired b reeding BALB/c m ice(M edina, 1982).
A lth o u g h the n u m b ers of m ice in early an d later generations w ere sm all the changes b e tw e e n the g e n e ratio n s are h ig h ly statistically significant. For all the m ice th ere w as a difference in transgene p o sitivity betw een the generations giving a v alu e of 9.097, w ith 2 degrees of freedom (df), p=0.01. G iven th at the p ro p o rtio n ap p e a rs to be declining, on looking for a trend, the Chi squared test for trend value is = 20.6, d f =1, p<0.001. This su g g ests th at there w as a difference betw een the different g en e ratio n s an d this difference w as progressive, occurring in a p articu lar direction.
In the eig h th g en e ratio n five d ifferen t m atin g p airs w ere estab lish ed b e tw e e n m ale an d fem ale m ice h eterozygous for the transgene. O f the 73 offspring from these pairs, 54 w e re tra n sg e n e p o sitiv e (74%) a n d 19 (26%) tra n sg e n e n e g a tiv e , close to th e ex p e c te d p ro p o rtio n s of 3 / 4 a n d 1 /4 , ex p e cted w ith h e te ro z y g o te -h e te ro z y g o te m atin g s. In co n tra st in m atin g s b etw e en h etero zy g o tes an d w ild -ty p e B A LB /c m ice there w ere few er transgene positive offspring th an the expected 50% an d som e w hole litters w ere tran sg en e negative. This is illu stra te d g raphically in Figure 3.9.
C h a p te r Three The N a tu r a l H is to n / o f the M o d e l 100% - | 90% - 01 80% — _> I 70%-4 60% -
I
I 50% ^ 40% £ S 30% H s e- 20% 10% — 0% i 'î 'i 'î 'î î m : : : :W :W > :¥ :¥ :■ P ' LV .V .V m S S S :::::::::: ::::::::::: .v.v.v I I I I 4 5 6 7 G enerationF igure 3.9 Percentage of transgene positivity by generation.
A ttem pts to increase tu m o u r incidence
O nce this colony h a d been established for ab o u t fifteen m o n th s, it becam e a p p a re n t th at a higher incidence of tum o u rs w as re q u ired to m ake the m o d el useful for th era p y experim ents. A n u m b e r of m an ip u latio n s w ere in itiate d to increase the incidence of tum ours. Some of these are still ongoing.
H om ozygotes
A hom o zy g o u s colony h as no w been established in the h o p e of a h ig h er incidence of tu m o u r d evelopm ent. As heterozygosity m ay be sufficient for the oncogenic effect of the transgene or at least m axim al en o u g h to p e rtu rb signalling th ro u g h neii a h ig h er incidence of tu m o u rs is n o t inevitable in h o m o zy g o u s mice. L ongterm experience of h o m o zy g o u s m ice in this colony is b a se d on the o ffsp rin g of a lim ited n u m b e r of h e te ro z y g o te -h e te ro z y g o te m a tin g s, p e rfo rm e d in th e se c o n d g e n e ra tio n . T w o hom ozygous m ice w ere identified w hen testcrossed w ith BALB/c m ales, on the basis of 18 a n d 16 tran sg en e positive offspring, respectively. B oth h av e d e v e lo p e d tu m o u rs - one a p ap illary m am m ary tu m o u r at th irteen m o n th s an d the o th er a ly m p h o m a at 21 m o n th s. A ge of o n set a n d ty p e of tu m o u rs d o n o t su g g e st a d ra m a tic effect of h o m o zy g o sity . The m ain p ra c tic a l a d v a n ta g e of a h o m o z y g o u s co lo n y is th a t it abrogates the n ee d for screening.
C h a p te r T hree______________________________________________________________T he N a tu r a l H is to r y o f the M o d e l
Identificatio n of h o m ozygous m ice from S outhern blots is only possible u sin g a single copy control an d d ensitom etry and involves a large n u m b e r of variables. C onsequently th ere is a w id e p o te n tia l m arg in of error. The only failsafe m e th o d is test-crossing w ith tra n sg e n e n eg a tiv e m ice. If a t least eig h t offsp rin g are tran sg e n e p o sitiv e the possibility of heterozygosity is 1 in 256 or less.
Using slot-blotting to determine zygosity
To e sta b lish w h e th e r slo t-b lo ttin g co u ld be u se d to ju d g e zy g o sity w e c o m p a re d subjective assessm ent of the density of slot blot w ells w ith test-crossing. This assum es sim ilar D N A y ield s from tailsn ip p re p a ra tio n s, sim ilar lo a d in g in w ells a n d th e n h o m o g en eo u s p ro b e h y b rid isatio n . A fter p ro b in g w ith the gene of in te re st the blo ts w ere p ro b e d w ith a housekeeping gene (p-actin) to confirm this hom ogeneity. G ro u p s of m ice w ith a p p a re n t 'light' an d 'd a rk ' w ells (greater th a n tw o fold difference) on slot b lo t h y b rid is a tio n h av e b een set u p a n d test-cro ssed a g a in st tra n sg e n e n e g a tiv e BA LB/c m ice. Based on a m inim um of 8 offspring all four m ice w ith 'd ark ' w ells w ho m a te d are h o m o zy g o u s. The fo u r m ice w ith 'light', b u t screen -p o sitiv e w ells, are h etero zy g o u s on testcrossing (Fisher's exact Test p=0.03).
H o m o zy g o u s m ice w ere established in the n in th generation an d initially d id n o t litter as freq u en tly as w h en heteozygous m ice w ere crossed w ith BALB/c mice. H o w e v er in gen eratio n s eleven an d tw elve the litter size and frequency is com parable w ith th a t in the earlier h etero zy g o u s m atings.
T he influence of litter n u m b er on tum our developm ent
In the first tw o h etero zy g o u s generations, m ice w ere m ated freely in o rd e r to ex p a n d the colony. T hereafter they w ere ro u tin ely m ated tw ice in o rd e r to place th em a t risk of tu m o u r d ev e lo p m e n t (B ouchard et al., 1989). In the fo u rth g eneration, the effect of litter n u m b e r w as assessed system atically. T w o g ro u p s - one of virgin m ice a n d one of m ice m a te d fo u r tim e s, w e re e s ta b lis h e d . T he o v e ra ll d iffe re n c e in tu m o u r d ev e lo p m e n t an d the difference in m am m ary tu m o u r incidence betw een the g ro u p s is o u tlin e d in Table 3.3 a n d F igure 3.10. The virgin m ice d e v e lo p e d few er m a m m a ry tu m o u rs (at 5.7% overall), th an those h a v in g at least tw o litters (22% overall). Those m ice m ated only once (only 10 mice) develo p ed no tum ours. It ap p e ars th at the risk of a m am m ary tu m o u r is n o t increased fu rth e r by four litters th an tw o (Table 3.3).
C h a p te r Three The N a tu ra l H is to n / o f the M o d e l