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BOR - Papers in Press, published online ahead of print July 7, 2004.
Biol Reprod 2004, 10.1095/biolreprod.104.029462
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BIOLOGY OF REPRODUCTION 72, 258–265 (2005)
DOI: 10.1095/biolreprod.104.029462
© 2005 by the Society for the Study of Reproduction, Inc.

Aberrant Gene Expression in Organs of Bovine Clones That Die Within Two Days after Birth1

Shijie Li3,4, Yanxin Li3, Weihua Du3, Lei Zhang3, Shuyang Yu3, Yunping Dai3, Chunjiang Zhao3, and Ning Li2

The State Key Laboratory for Agrobiotechnology in Livestock and Poultry,3 China Agricultural University, Beijing 100094, China Department of Biochemistry and Molecular Biology,4 College of Life Science, Hebei Agriculture University, Baoding 071002, China


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cloning by somatic nuclear transfer is an inefficient process in which some of the cloned animals die shortly after birth and display organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we used real-time quantitative reverse transcription-polymerase chain reaction to examine expression patterns of eight developmentally important genes (PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF) in six organs (heart, liver, spleen, lung, kidney, and brain) of both cloned bovines that died soon after birth (n = 9) and normal control calves produced by artificial insemination. In somatic cloning of cattle, fibroblasts have often been used for doner nuclei, and the effect of the age of the fibroblast donor cells on gene expression profiles was investigated. Aberrant expressions of seven genes were found in these clones. The majority of aberrantly expressed genes were common in clones derived from adult fibroblast (AF) and in clones derived from fetal fibroblast (FF) compared to controls, whereas some genes were dysregulated either in AF cell-derived or in FF cell-derived clones. For the studied genes, kidney was the organ least affected by gene dysregulation, and heart was the organ most affected, in which five genes were aberrant. Most dysregulations (12 of 19) were up-regulation, but PDGFRa only showed down-regulation. VEGF, BMP-4, PCAF, and Hsp70.1 were extremely dysregulated, whereas the other four genes had a low level of gene dysregulation. Our results suggest that the aberrant gene expression occurred in most tissues of cloned bovines that died soon after birth. For each specific gene, aberrant expression resulting from nuclear transfer was tissue-specific. Because these genes play important roles in embryo development and organogenesis, the aberrant transcription patterns detected in these clones may contribute to the defects of organs reported in neonatal death of clones.

developmental biology, early development, gene regulation, growth factors, stress


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several mammalian species have been successfully cloned by somatic cell nuclear transfer (NT) [1–7], but only a small proportion of the embryos produced using adult or fetal somatic cells develop into living young (typically between 0 and 4%) [8]. Clearly, the causes of low efficiency must be identified if the medical and agricultural potentials of cloning procedures are to be realized. In cloning mammals by somatic cell nuclear transfer (NT), the doner nucleus requires epigenetic reprogramming to a totipotent ground state [9]. The incomplete reprogramming of donor somatic cell nuclei leading to aberrant (or even lack of) expression of some developmentally important genes has been implicated as a primary reason for this low efficiency [10].

Some genes important for early embryogenesis have been studied in cloned preimplantation embryos [10, 12]. Examination of the expression of many imprinted genes in cloned mice that survived to birth has shown that some of them are abnormal [13, 14]. Many cloned offspring die shortly after birth and often exhibit phenotypic abnormalities, but the reasons for these abnormalities have not been clarified. One objective of the present study was to investigate the expression of developmentally important genes in the organs of cloned bovine of neonatal death to determine the possible genetic causes of the neonate's death and of the organ abnormalities in animals produced using NT techniques.

The X-inactive specific transcript (Xist) plays a crucial role during X-chromosome inactivation (XCI) [15]. Hsp70.1 (70-kDa heat-shock protein) is a stress protein involved in facilitating protein folding and assembly as well as in stabilizing damaged proteins for repair and degradation in cells [16]. PCAF (p300/CFBP-associated factor), which has histone acetylase activity, is involved in cell-cycle arrest, transcriptional activation, plays a role in transcriptional activation, cell-cycle arrest, and cell differentiation in cultured cells [17]. Vascular endothelial growth factor (VEGF) [18, 19], platelet-derived growth factor receptor {alpha} (PDGFRa) [20–23], bone morphogenetic protein 4 (BMP4)[24–29], fibroblast growth factor receptor 2 (FGFR2), and fibroblast growth factor 10 (FGF10) [30–36] play important roles in embryo development and organogenesis. In the present study, the transcripts of Xist, FGFR2, PDGFRa, Hsp70.1, VEGF, BMP4, and FGF10 were examined in tissues from both deceased neonatal clones and normal control calves.

Cloning is a multistep procedure, and many factors may affect cloning efficiency. The donor cell type, age, and cell-cycle stage are factors generally thought to affect cloning efficiency in mammals. In somatic cloning of cattle, many kinds of differentiated cells have been tested, but most investigators have used skin fibroblasts as donor nuclear material. Fibroblasts offer several advantages for future applications of somatic cloning. First, a skin biopsy specimen can be easily obtained from a valuable animal, so the production of large numbers of genetically identical copies of the animal are possible. Second, fibroblasts can be easily cultured and stored frozen, so they probably are good candidates for genetic modification to produce transgenic animals. Age of the fibroblast donor cell has no effect on in vitro development of bovine NT embryos [37], but other reports indicate that clones derived from adult cells frequently abort during the later stages of pregnancy and that calves developing to term show a higher number of abnormalities than those derived from newborn or fetal cells [38]. In the present study, nine deceased cloned bovines were derived from adult fibroblast (AF) and fetal fibroblast (FF). The other objective of the present study was to examine the effect of donor cell age on the expression of developmentally important genes in cloned bovines.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Nuclear Transfer

The NT procedures have been described in detail by Gong et al. [39]. The donor nuclei were obtained from skin fibroblast cells of elite Holstein cow (age, 4 yr) and from FF cells of a female fetus (gestational age, 40 days). Approximately 27.9% (n = 283) and 37.9% (n = 294) of reconstructed embryos derived from AF and from FF, respectively, developed into blastocysts, with 11 (14.9%, n = 74) and 10 (22.7%, n = 44) of the transferred embryos, respectively, developing into full-term calves. Six of the 11 calves from AF cells (ww, bw, cw, sw, yw, and lw) survived and remained healthy, and five (AF1–AF5) died. Six of 10 calves from the FF cells (dw, qq, tt, jm, xw, and aw) survived and remained healthy, and four (FF1–FF4) died. All of the deceased cloned cattle died within 48 h of birth.

Tissue Collection

The cloned animals were dissected immediately after death, and normal control calves produced by artificial insemination were killed within 48 h after birth. Samples of all major internal organs of deceased cloned calves and normal control calves were immediately collected and frozen in liquid nitrogen for later analysis.

Preparation of RNA

We extracted total RNA from organ samples of the deceased cloned calves and normal control calves using a TRIZOL RNA isolation kit (Invitrogen). The RNA preparations were treated with RNase-free DNase I to remove possible contaminating DNA and were stored at –70°C.

Reverse Transcription

Reverse transcription (RT) was done using an RT kit (Promega) with approximately 1 µg of RNA in a total volume of 20 µl. The RT reaction was conducted according to the manufacturer's guidelines using oligo(dT) primers and AMV reverse transcriptase enzyme in a volume of 20 µl to prime the RT reaction and produce cDNA. Tubes were heated to 65°C for 5 min to denature the secondary RNA structure. The RT reaction was completed by adding 5 U of Superscript RT enzyme (Promega) before incubation at 37°C for 1 h and then 70°C for 15 min.

Quantitative RT-Polymerase Chain Reaction

The quantification of all gene transcripts was carried out by real-time quantitative RT-polymerase chain reaction (PCR).

Gene expression levels were measured using a DNA Engine Opticon 2 fluorescence detection system (MJ Research) and DyNAmo SYBR Green qPCR kit (MJ Research). SYBR Green I was a double-stranded, DNA-specific fluorescent dye. The sequences of the PCR primers used for RT-PCR quantification are shown in Table 1. The PCR reaction mixture (20 µl) contained 10 µl of DyNAmo SYBR Green qPCR mix, 5 µl of primer (0.3 µM forward and 0.3 µM reverse), and 5 µl of cDNA template (<10 ng/µl). The PCR protocol included uracil-N-glycosylase (UNG) enzyme incubation at 50°C for 2 min and an initial denaturation at 95°C for 10 min. This was followed by 40 cycles of 10 sec each at 94°C for DNA denaturation, 20 sec at different temperatures for annealing of primers, 20 sec at 72°C for primer extension, and 1 sec at a different elevated temperature for data acquisition. The annealing and data acquisition temperatures of primers are shown in Table 1. The reannealing step (at 72°C for 10 min) allowed reformation of fully duplexed DNA in performing the agarose gel analysis. The melting protocol called for heating from 65 to 95°C, holding for 1 sec at each temperature, with increases of 0.2°C per step


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  		TABLE 1. Primer and cycle parameter used for real-tine RT-PCR

All samples were measured in triplicate. Performing the data-acquisition step at an elevated temperature was helpful in minimizing the interference of primer-dimers with the quantification. Fluorescent data were acquired during the data-acquisition step. The melting curve analysis was used to check the specificity of an amplified product. Amplified product underwent electrophoresis on agarose/ethidium bromide gels and was visualized under ultraviolet (UV) light. In addition, product identity was confirmed by sequencing. The PCR products were purified using DNA wizard cleanup kit (Promega) and directly sequenced by Taq cycle sequencing using DyeDeoxy terminators in an automated sequencer (ABI 377; Applied Biosystems).

Expression was quantified by the relative standard curve method. The quantification was normalized to an endogenous RNA control Glyceraidehyde-3-phosphate dehydrogenase (Gapdh, a housekeeping gene), and standard curves were plotted for each target and the endogenous genes. Each of the cDNA fragments of the target gene was purified using DNA wizard cleanup kit and cloned into plasmids for use as standards in quantifying gene expression level. A standard graph of the cycle threshold (CT) values obtained from serial dilutions (10–104 copies/well) of the plasmid was created. Fluorescence was acquired in each cycle to determine the CT or the fluorescence baseline at which fluorescence rose above background for each sample. The Opticon Monitor 2 software produced a best-fit fluorescence baseline and the standard graph. For each experimental sample, the amounts of mRNA of each target gene and Gapdh were determined from the CT plotted on the respective standard curves. The mean values of the replicate wells run for each sample were calculated; subsequently, the mean quantity of each target gene was divided by Gapdh to obtain a normalized value for each transcript.

Because the experimental materials were the tissues with many serious aberrations, another endogenous RNA control ß-actin was used to identify the research results of the relative expression levels of target genes normalized by Gapdh.

Statistical Analysis

Data were analyzed using SPSS (version 10.0; SPSS, Inc., Chicago, IL). An independent sample t-test was used to analyze differences in mRNA expression assayed by quantitative RT-PCR between the clones and the normal controls or between the AF-derived animals and the FF-derived clones. Differences were considered to be statistically significant at P < 0.05.

Animal Care

These studies were performed at the University of China Agriculture (Beijing, China), and all procedures were in accordance with the Guiding Principles for the Care and Use of Laboratory Animals.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
All deceased cloned cattle died within 48 h of birth, and all had aberrations in many of their organs. The principal abnormalities at necropsy are shown in Table 2. Seven hearts from deceased clones were abnormal, displaying compensatory cardiac hypertrophy, vascular incompetence and patent foramen ovale, cardiac hemorrhage, and necrosis. At birth, atelectasis was observed in the lungs of five cloned calves. Other common abnormal characteristics included thickening of the lung alveolar wall, pulmonary hemorrhage, inflammation, and congestion. Hepatomegaly and congestion were found primarily in livers of cloned calves. Oddly, AF5 had six lung lobes, which did not connect to each other (Fig. 1E), and the kidney of FF2 had much fat, both inside and outside (Fig. 1C). AF4 had only a small amount of brain tissue, with necrosis, hemorrhage, and edema (Fig. 1A). The heart of AF3 was enlarged (0.4 kg) and had a patent foramen ovale (Fig. 1B). A large cyst was found on the liver of AF1 (Fig. 1F). Figure 1D shows the heart of AF1, with cardiac muscle putrescence and congestion. These findings suggest serious aberrations in the organs of cloned bovines that die soon after birth.


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  		TABLE 2. Principal abnormalities at necropsy of deceased cloned cattle



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  		FIG. 1. Pictures of representative abnormalities found in cloned calves that died soon after birth. A) Brain of AF4, with only a small amount of brain tissue and with necrosis, hemorrhage, and edema. B) Heart of AF3, of an enlarged size (0.4 kg) and with a patent foramen ovale. C) Kidney of FF2, with fat inside and outside. D) Heart of AF1, with cardiac muscle putrescence and congestion. E) Lung of AF5, with six lung lobes that do not connect and with congestion. F) Liver of AFI, with a large cyst

Amplification products were identified by melting curve profile analysis and confirmed by gel electrophoresis and by sequencing. Figure 2 shows photographs of representative gels from real-time RT-PCR analysis of 420 base pairs (bp) of Gapdh, 398 bp of ß-actin, 226 bp of Xist, 340 bp of VEGF, 425 bp of Hsp70.1, 239 bp of FGF10, 220 bp of PCAF, 220 bp of FGFR2, 249 bp of PDGFRa, and 223 bp of BMP4. The final relative abundances of each target gene normalized by Gapdh were the same as the results normalized by ß-actin.



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  		FIG. 2. Photographs of representative gels from real-time RT-PCR analysis. Lane 1: 398 bp of ß-actin; lane 2: 223 bp of BMP4; lane 3: 249 bp of PDGFRa; lane 4: 220 bp of PCAF; lane 5: 240 bp of VEGF; lane 6: 220 bp of FGFR2; lane 7: 425 bp of Hsp70.1; lane 8: 226 bp of Xist; lane 9: 420 bp of Gapdh; lane 10: 239 bp of FGF10

Gene expression in each of the two types of clones was first compared with that of controls. The relative transcription abundances of PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF in liver, heart, lung, spleen, kidney, and brain of cloned calves from both types of donor cells as compared to controls are shown in Table 3. Aberrant expressions of genes were found in all studied tissues. The majority of aberrantly expressed genes were common to both types of clones, whereas some were dysregulated either in AF-derived or in FF-derived clones. In heart, five abnormally regulated genes were found, in which VEGF (P < 0.05), BMP4 (P < 0.01), and PCAF (P < 0.05) were elevated in both types of clones compared to controls, whereas FGF10 showed a higher level (P < 0.05) only in FF-derived clones and Xist a higher level (P < 0.01) only in AF-derived clones. In brain, a higher level of VEGF (P < 0.01) and a lower level of Hsp70.1 (P < 0.01) were observed in both types of clones, but BMP4 was elevated (P < 0.01) only in AF-derived clones. In kidney, a lower expression level of PDGFRa (P < 0.01) was seen in both types of clones, but FGF10 showed a lower level (P < 0.05) only in FF-derived clones. In spleen, BMP4 was reduced noticeably (P < 0.01), and VEGF was elevated (P < 0.05), in the two types of clones; however, PCAF fell (P < 0.05) only in AF-derived clones. Three genes had aberrant expression in lung: Hsp70.1 (P < 0.01) and BMP4 (P < 0.05) were elevated in both types of clones, and PDGFRa was reduced (P < 0.05) in FF-derived clones. For liver, PCAF (P < 0.05) was elevated in both types of clones, and Hsp70.1 showed a higher level (P < 0.01) only in AF-derived clones.


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  		TABLE 3. Relative transcript levels of genes in deceased calves derived from two kinds of donor cells.a

In addition to comparing the gene expression of two types of clones with those of controls, we compared the gene expression between the two types of clones with each other. The expression of only four genes showed a significant difference between AF-derived and FF-derived clones. The expressions of BMP4 in brain, PDGFRa in lung, and Xist in heart showed a higher level (P < 0.01) in AF-derived clones than in FF-derived clones. Conversely, FGF10 in heart showed a lower level (P < 0.05) in AF-derived clones than in FF-derived clones.

Although the difference in the expression of some genes between the clones and the normal controls did not reach statistical significance in some organs, the variability among the individual clones was noticeable. The expression of Hsp70.1 showed noticeable variation in heart, liver, spleen, and kidney in the clones (Fig. 3A). For example, the expression of Hsp70.1 in the lung of FF3 and the kidney of FF1 was approximately sevenfold higher than in controls, but the expression of Hsp70.1 was not detected in the heart of either FF1 or FF2, in the spleen of either AF4 or AF5, or in the liver of either FF2 or FF3. In the case of FGF10, wide variations were also observed in most studied tissues from the dead cloned calves (Fig. 3B). In addition to no expression of FGF10 in the spleen and liver of FF2, very low expression levels were observed in the lung of both FF2 (0.003) and FF3 (0.006) and in the spleen of both FF1 (0.09) and AF3 (0.11) compared to the controls; however, a high level was observed in the heart of FF2 (6.61-fold), the liver of AF5 (5.73-fold), and the brain of AF5 (5.37-fold). Expression of BMP4 in the spleen of FF3 and of PDGFRa in the heart of AF3 was absent. The expression variations of Xist and FGFR2 in deceased cloned animals were smaller in eight candidate genes.



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  		FIG. 3. The relative transcript levels of Hsp70.1 (A; in heart, liver, spleen, and kidney) and FGF10 (B; in heart, liver, spleen, lung, kidney, and brain) of deceased calves derived from two kinds of donor cells—AF (n = 5) and FF (n = 4)—compared with controls (n = 3, normalized to 1), which are represented by the dashed line

Results of all the dysregulated genes, according to the organ studied, are summarized in Table 4. For the studied genes, kidney was the organ that was least affected (two genes) by gene dysregulation, whereas heart was the organ that was most affected (five genes). Most cases of dysregulation (12 of 19) were up-regulation, but PDGFRa only showed down-regulation. VEGF, BMP-4, PCAF, and HSP were extremely dysregulated, whereas the other four genes had a low level of gene dysregulation.


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  		TABLE 4. All the dysregulated genes according to the organ studied.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The efficiency of animal production using somatic cell NT is very low. The doner nucleus must establish normal expression patterns to ensure that the reconstructed embryo develops successfully. To determine the possible genetic causes of death in animal production using NT techniques, the transcription of eight genes (Xist, VEGF, Hsp70.1, FGF10, PCAF, FGFR2, PDGFRa, and BMP4) was compared in the major internal organs of deceased clones and normally reproduced control calves using real-time RT-PCR. These genes are known to have important functions during development of the embryo and organogenesis in mammals.

In female mammals, dosage compensation is gained by X-chromosome inactivation, with random transcriptional silencing of one of the two X chromosomes in female cells during late blastocyte development, and the Xist, (X-inactive-specific transcript) plays a crucial role for XCI [40]. Previously, patterns of XCI have been reported to be normal in cloned mice [41], but in the present study, expressions of Xist in the heart of AF-derived clones was higher than in the controls. In addition, overexpressions of Xist were found in cloned bovine embryos [42], and hypomethylation of Xist was observed in deceased cloned bovines [43]. Overexpression of Xist in the hearts of clones that died soon after birth may result in aberrant reactivation of the silent X chromosome, where incomplete nuclear reprogramming may generate abnormal epigenetic marks on the X chromosomes and then affect the normal patterns of XCI.

In the present study, aberrant expression of Hsp70.1 was observed in the lung, brain, and liver of cloned animals. Heat shock proteins are stress proteins, and their binding of apoptosis-inducing factor (AIF) inhibits the nuclear import of AIF [44] and, therefore, protects the embryo from undergoing apoptosis [45]. The transcripts of Hsp70.1 were not detected in bovine blastocysts from NT [12]. The present results suggest that the NT embryos did not copy in the adverse environment. Consequently, the normal expression of Hsp70.1 was affected, disrupting the viability of offspring from the NT.

Both DNA methylation and histone acetylation on nuclear chromatin play important roles in the regulation of gene activation, where histone acetylation is involved in the inheritance of cell memory [46]. Several reports have shown inefficient demethylation and inappropriate re-establishment of DNA methylation during somatic NT. PCAF is a transcriptional coactivator with intrinsic histone acetylase activity. It contributes to transcriptional activation by modifying chromatin and transcriptional factors [47, 48] in addition to its role in transcriptional activation, cell-cycle arrest, and cell differentiation in cultured cells. In the present study, aberrant expression of PCAF was observed in some organs of cloned bovines, which agrees with the research of Enright et al. [49], who concluded that histone acetylation status is remodeled in cloned bovine embryos. They also found that histone deacetylase inhibitor and DNA methyl-transferase inhibitor can improve the reprogramming ability of donor cells [50]. Therefore, NT leads to epigenetic abnormalities that affect the normal expression of PCAF.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, has been implicated in the regulation of blood vessel formation (i.e., vasculogenesis and angiogenesis). Development of the cardiovascular system depends on the generation of precise VEGF concentration gradients. A decrease in the amount of VEGF produced during development of the embryo may lead to decreased angiogenesis, with fatal consequences [18, 19]. VEGF modulates early heart valve formation [51]. Significantly higher expression of VEGF was seen in cloned animal heart, spleen, and brain compared to controls. Coincidentally aberrant valvulogenesis was seen in cloned heart, and congestion and hemorrhage were seen in some cloned heart, brain, and spleen. The aberrant expression of VEGF may contribute to these defects as reported in the clones that died soon after birth.

In the cloned animals, a significantly lower expression of PDGFRa was observed in the kidney and lung compared to controls. PDGFRa plays an important role in lung development [21, 22] and has a relationship with renal development and disease [23, 52]. The results suggest that NT leads to aberrant epigenetic activity affecting the normal expression of PDGFRa, particularly in the lung and kidney of cloned bovines.

BMPs, a subgroup of the transforming growth factor-ß superfamily, have been found to play roles in many embryonic patterning events [53] and in all aspects of embryonic development and organogenesis [24]. In the present study, the expression of BMP4 showed a higher level (P < 0.01) in cloned lungs compared to control calves. Aberrant morphogenesis was found in the lungs of seven clones. Both AF5 and FF2 showed very poor lung morphosis: AF5 had six lung lobes, which did not connect to each other, and FF2 had only one lung lobe. Previous researchers showed that BMP4 was one of the key growth factors essential for lung development [25] and that overexpression of BMP4 caused abnormal lung morphogenesis, with cystic terminal sacs and inhibition of epithelial proliferation [54]. Therefore, aberrant expression of BMP4 may contribute to lung abnormalities in cloned animals. Proper septation and valvulogenesis during cardiogenesis depend on interactions between the myocardium and the endocardium. BMP4, as a signal from the myocardium, directly mediates atrioventricular septation. The endocardial cushion, cardiac valve, and semilunar valve maturation are controlled by BMP-signaling pathways [55, 56]. The normal expression of BMP4 plays a major role in the atrioventricular septation of the mouse heart [26]. In the present research, the expression of BMP4 showed a higher level (P < 0.01) in cloned hearts with valvular incompetence, patent foramen ovale, or enlarged right ventricle, which suggests that aberrant expression of BMP4 may also contribute to the heart abnormalities of cloned animals.

In cloned bovines, age of the fibroblast donor cell has been reported to have no effect on the in vitro development of bovine NT embryos [37], but other reports indicate that clones derived from adult cells frequently abort during the later stages of pregnancy and that calves developing to term show a higher number of abnormalities than those derived from newborn or fetal cells [38]. The present results suggest that the age of different donor cells affected the gene expression in cloned bovines, which is consistent with the results of NT experiments in the mouse [9]. Many other genes are involved in mammalian embryonic development and organogenesis, and further research concerning these genes will increase our understanding of nuclear reprogramming events following somatic cell NT, embryonic development, and organogenesis.


   FOOTNOTES
 
1 Supported by the State High-Tech Research & Development Program of China and Natural Scientific Foundation of Beijing. Back

2 Correspondence: Ning Li, The State Key Laboratory for Agrobiotechnology in Livestock and Poultry, China Agricultural University, Yuanmingyuan West Road 2, Beijing 100094, China. FAX: 010 62893904; ninglbau{at}public3.bta.net.cn Back

Received: 9 March 2004.

First decision: 2 April 2004.

Accepted: 21 May 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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