J. of Aquaculture Vol. 19(4) : 281-287, 2006 µ Journal of Aquaculture Korean Aquaculture Society ã(misgurnus mizolepis) : II. sƒ û«*, 1, y 2,, ½ w w/wxy, 1ww wœw, 2 w w Intraspecific Androgenesis in Mud Loach (Misgurnus mizolepis): II. Diploid Restoration and Viability Assessment Yoon Kwon Nam*, In-Chul Bang 1, Choong Hwan Noh 2, Young Sun Cho and Dong Soo Kim Department of Aquaculture & Institute of Marine Living Modified Organisms (imlmo), Pukyong National University, Busan 608-737, Korea 1 Department of Marine Biotechnology, Soonchunhyang University, Asan, 336-745, Korea 2 Marine Resources and Research Department, Korea Ocean Research and Development Institute, Ansan 426-744, Korea Intraspecific diploid androgenesis was achieved in mud loach (Misgurnus mizolepis) by the inhibition of the first mitotic division using combined thermal treatment. A combined thermal treatment (heat shock at 40.5 o C for 120 sec followed by cold treatment at 1 o C for 45 min) applied to the 1st metaphase of cell division (28 min post insemination at 25 o C) successfully recovered viable androgenetic diploidy. Mean hatching success of the androgenetic diploid group was 29.6%, and the average yield out of total eggs taken was about 7% assessed at 1 week of age. However, relatively large variations in the yield of diploid androgenesis were observed among different egg batches used as cytoplasmic donors. Successful diploidization was confirmed by flow cytometric analysis, and parthenogenic reproduction in a paternal exclusive manner was verified with transgene dosage. Significant mortality was found in most androgenetic groups especially from hatch to 1 month of age, although such mortality was stabilized later. Keywords: Diploid androgenesis, Mud loach, Parthenogenic reproduction, Viability 1ùw»(blocking of the first mitotic division) œ (induced artificial parthenogenesis) 4 (genome duplication; tetraploidy) w» ù w 1s» 2s» (embryonic development) jš, (chromosome set)ƒ ƒ(doubling) w»(thorgaard, 1986; Arai, 2001). p (induced androgenesis) k (loci), xw(homozygous)yw š mw p x šw (Bercsenyi et al., 1998; Kirankumar and Pandian, 2003). *Corresponding author: yoonknam@pknu.ac.kr 281» š m wš w, p (genome-restoration) xw w r ƒ yš (Zhang et al., 2000; Babiak et al., 2002a; Kirankumar and Pandian, 2004; David and Pandian, 2006). ù z û 1) yù ù w w, 2) ù yy ùy RNA q w, 3) w s w (nuclearcytoplasmic instability, NCI) š 4)»x û»»w(bercsenyi et al., 1998; Babiak et al., 2002b). s mw
282 û«,, y,, ½ w { û šš (Bongers et al., 1994; Babiak et al., 2002b). z l w» w, ù w (intraspecies androgenesis) œ w tƒ, ù w NCI z yw x w w k., yy ù 1ùw wš, 1ùw w š w zƒ y z» z yw sƒ w w w ƒ y w. ù š ã (Misgurnus mizolpis) ã (interspecies androgenesis) û z( 4%; Nam et al., 2002) w» w y ü ã» wš w. w ã xy t, intraspecies haploid androgenesis yw (Nam et al., 2006), 1ùw mw ã wš» z» sƒwš w. x I: ù yy mw x ã w w w 5 e s³ (body weight BW) 45Û 13 g f 15Û5 g f e w. el Kim et al. (1994) k y(hcg) w ww. w ù yw. 8 fl ù j»» ù 4 f w, 5 f yww x w. ù yy j» w Nam et al. (2006) w (UV) ww. UV 10,800 ergs/mm 2 w yy ù 3,000 50 µl (1:30 in saline), mw œ w. 25 o C w ww 1ùw» 28 z Nam et al. (2004) w š (40.5 o C 2) (1 o C 45) ww ww. 1ùw ƒ óù 25 o C w y¾( z 24~27 ) w. z sƒw» w ƒ 3 w, y ù y»(y z 36 )¾» w. x II: f e z w ã f el ù ywš ù e w w. wš m w yw 16 f( 36~61 g)l œ» mw ƒƒ ù ywš» ù yy w. œ w w 12 fl yw w. ƒ w ƒ 3,000Û120 ù swm w. œ,, y, y»» x I w wwš, ƒ x 3 y w z w f e z w. x III: sƒw» w ùy zl» z» w. x I II k 3 f 3 fl w wš ùy ƒ 1, 2, 3 4» w. z 1 y 4 ¾ z» 3z w. ùy 36 ƒ ƒ 50 L 3 w w 25Û1 o C wš k x Kim et al. (1994) w. x IV: flow cytometry z yw» w flow cytometry w ã s DNA w dw. e homogenization (10 mm Tris ph 7.8, 5 mm EDTA ph 8.0, 50 mm NaCl) w z 20 µm mesh w s zw. z s 50 µg/ml propidium iodideƒ sw (1x PBS ph 7.6, 0.01% NP 40) 1 w z WinBryte HS flowcytometer (BioRad, USA) w s s³ DNA w w., ã (1.4 pg/cell), š x
s(2.8 pg/cell) w. y 2 w l x s w homogenization» w flow cytometry w. x V: t w œ xy t w, duplication yw. x w xy chloramphenicol acetyltransferase (CAT) reporter construct xw(homozygous) xy m w w p Nam et al. (1999, 2000)». x I k xy f l 3 fl yy ù» w y 23 flow cytometry mw yw y ƒ 12 transgene dosage w. ƒ (fin)l Nam et al. (1999) w genomic DNA w genomic DNA 2 µg dot blot hybridization w. genomic DNA w positively charged nylon membrane (Roche, Germany) dot blot apparatus (Bio- Rad) w 3 spotting w CAT-transgene probe (Nam et al., 2000) w hybridization w. Hybridization sww stringent washing signal detection (DIG Non-radioactive DNA Labeling and Detection kit, Roche) instruction manual w ww. Internal control ã growth hormone gene probe (Nam et al., 2002) w CAT transgene signal normalizationw. yw» w (doubled transgene dosage; homozygous at transgene loci) bi-parental (heterozygous transgene dosage) signal image analysis software Quantityone (Bio-Rad) w. m x I z sƒ w ã 283 w y» x (ANOVA) Duncan s multiple range test w P<0.05 sƒ w. x III student s t-test mw wš(p<0.05), x V transgene dosage Student s t-test w bi-parental transgene signal (P<0.05) w. š w x I w ù z s³ 85% ùkù(figures not shown) w Nam et al. (2006) w z UVƒ. 1ùw» mw w, 90% y 10,800 ergs/mm 2 UV 51.4% s³ y, š UV ƒ w 29.6% s³ y ù kü(table 1). yz» s. y s³ 87.5%ƒ ùy»¾ wù 37.5% š ù ùy w w sw. k 1ùw w w w ƒ qù(pandian and Koteeswaran, 1998) ù potential aneuploidy hypodiploidy x, w r w s w w (Corley-Smith et al., 1996; Maremgpmo and Onoue, 1998; Tanck et al., 2001). xwyw» x(recessive traits)» w w q(lin and Dabrowski, 1998; Paschos et al., 2001). y 1 Table 1. Percent hatching, early survival, incidence of 2N and yield of intraspecific androgenesis in mud loach 1 Genotype Hatching success (%) 2 Early survival rate (%) 2 Incidence of diploidy (%) 3 Normal diploid control 91.2±5.8 a 87.5±6.8 a 100±0.0 a Androgenetic haploid 51.4±9.2 b - 7.1±2.7 b Androgenetic diploid 29.6±8.1 c 37.2±5.4 b 98.1±2.1 a 1 Mean±SDs are based on at least three replicate examinations. Means with same letters within a column did not differ significantly at P=0.05 based on ANOVA. 2 Hatching success and early survival up to yolk sac absorption were estimated as percentages of eggs inseminated and hatched larvae, respectively. 3 Ploidy estimation was carried out using flow cytometry with 1-week-old fish for diploid groups, while the score for androgenetic haploid group was based on the examination of external morphology at 1 day post hatching.
284 û«,, y,, ½ l 3z w 54 flow cytometry ww aneuploidy peak 1 wš 53 (98.1%) yw DNA w y z ùkû (Table 1; peaks not shown). ù 1ùw w ƒ 7.1% w ƒ w wš. û xw w UVƒ 100% ù yy j w» w k (Maremgpmo and Onoue, 1998; Tanck et al., 2001; Nam et al., 2006). Cytoplasmic donor z» x k w f el ù(cytoplasmic donor) w z w. (yield) w sƒw w w š w ù ùkü. (yield) = [y(%) ùy» ¾ (y w ) ùy»l y 1¾ (%) x (y 1 flow cytometry ; group 1220 )] x w f e y 5894% w ùkû(histograms in Fig. 1a) ƒƒ w ù (egg batch) w ww, ù w 0.05% (f no. 5) 10.3% (f no. 3)¾ w egg batch j ùkü(line drawing in Fig. 1a). x y, y û z w w ƒ. Fig. 1b mw» y (X ) w ƒ e(y ) txw, Fig. 1b y 70% w œ ƒ w ùkû. w y 70% y y w ùkü, 7% y» w y 90% w ƒw (Fig. 1b). w e Fig. 1. Maternal influences on the yield (%) of diploid intraspecific androgenesis in mud loach. UV-inactivated egg batches from 16 independent females were separately inseminated with the pooled sperm from 9 males, and subjected to thermal treatment for blocking the 1st cleavage. a) Yields (mean±sds based on triplicate examinations per cross) of diploid androgenesis at 1 week of age (line drawing) addressed for each female with the comparison of percent hatching for the non-treated control group (histograms) from the same female. b) Percent yields represented as function of the hatching success in diploid control groups without data separation into each female group. Details for calculating the yield of androgenesis can be referred to the equation in Materials and Methods. m ù j w x wš (Bongers et al., 1995), p y w w ù w w wš. ã ùy z w, w û (Table 2). ùy z s ùk ü p sƒ»» w ùkü. ùy z 1, s³ 97.2%
ã 285 Table 2. Percent survival of androgenetic diploid mud loaches along with normal diploid controls as function of age Age Normal diploid control Androgenetic diploid 1 week 98.1±1.6 a 69.4±7.3 a 2 week 95.4±1.6 a 54.6±5.8 a 3 week 91.7±2.8 a 48.1±4.2 a 4 week 88.9±2.8 a 38.9±7.3 a 2 month 85.2±1.6 a 37.0±4.2 a 3 month 84.3±3.2 a 34.3±4.2 a 4 month 83.3±2.8 a 34.3±4.2 a Fish were allocated into replicate tanks after yolk sac absorption (see Materials and methods). Means with same letters within a row were not significantly different at P=0.05 based on student s t-test. û 69.4% ùkü(p<0.05). z 2, 3 4¾ ƒ 6.5~14.5% sƒ ƒ wš, y 1 s 12% 61% w e ùkü. wš y 1 zl y w ùkü y 4 ¾ s(0~2.8%) š s(0~3.7%) m ùkü (P>0.05). ù ùy l y 4 ¾ (34.3%) (83.3%) 41% ww ùkû(table 2). e x ù» w šwš w w ù kü(bercsenyi et al., 1998; Babiak et al., 2002b). w m w xw z xw k tx (recessive genes) x wy» x q p», e k s 1ùw w z(adverse effect)ƒ y z» ùküš. ƒw, š ƒ y z» w w öe ã k š (Thorgaard, 1986; Kim et al., 1994).» w w š» z» x ùkù š m ƒ vw š. Transgene dosage w xy t w m w y( duplication) w. xw CAT-transgenic Fig. 2. Transgene dosage assessment in androgenetically derived homozygous (Andro-2N) and bi-parental hemizygous (Bi-parental 2N) mud loaches. a) Representative dot blots showing the difference in hybridization signal between homozygous and hemizygous fish. b) Densitometric analysis of the hybridized signals using the GH gene probe as a normalization control. Statistical difference was found between androgenetic diploid and bi-parental diploid groups based on student s t-test (P<0.05). fl f(non-transgenic)l yy ù w y z 2~3 z CAT xy w x y s copy (transgene dosage) ƒ(homozygous y) w(fig. 2). e w w (bi-parental 2N)l ƒ 12 w dot blot hybridization xy x w xy f xw k wš (photographs not shown). ù xy ùkû (chromosome-set) w z (Fig. 2a). y mw x y xy xw xk ùkü xy f f w transgenic hemizygous k ùkü (see Nam et al., 2002). GH gene ü w CAT-xy normalizationw
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