Selection of in vitro produced, transgenic embryos by nested PCR for efficient production of transgenic goats.

The production of valuable pharmaceutical proteins using transgenic animals as bioreactors has become one of the goals of biotechnology. However, the efficiency of producing transgenic animals by means of pronuclear microinjection is low. This may be attributed in part to the low integration rate of foreign DNA. Therefore, a large number of recipients are required to produce transgenic animals. We recently developed a transgenic procedure that combined the techniques of goat oocyte in vitro maturation (IVM), in vitro fertilization (IVF), microinjection, preimplantation selection of the transgenic embryos with nested PCR and transferring the transgenic embryos into the recipient goat uterus to produce transgenic goats. Thirty-seven transgenic embryos determined by nested PCR were transferred to thirty-two recipient goats. In the end, four live-born kids were produced. As predicted, all the live kids were transgenic as identified by PCR as well as Southern blot hybridization, The integration rate was 100% (4/4) which was completely in accordance with the results of embryo preimplantation detection. The results showed a significant decrease in the number of recipients required as only 8 recipients (32/4) were needed to obtain one live transgenic goat. We suggest that the transgenic system described herein may provide an improved way to efficiently produce transgenic goats on a large scale.

Reversal of aberrant splicing of beta-thalassaemia allele (IVS-2-654 C-->T) by antisense RNA expression vector in cultured human erythroid cells.

The antisense fragment targeting the aberrant splice sites of the beta-thalassaemia allele, IVS-2-654 C-->T (beta654), pretranscript was cloned into the mammalian expression vector, pcDNA3. The recombinant construct, pCMVA, was then used to repair the defective splicing of the beta654 mutant pretranscript in cultured beta654 erythroid cells by the lipofectin-mediated DNA transfection method. The total RNA was extracted at given time points after transfection and the effect of antisense RNA was studied by reverse transcription polymerase chain reaction (RT-PCR)-mediated mRNA quantitative assay, as well as globin chain microbiosynthesis. The antisense fragment transcribed from pCMVA effectively improved the beta654 splicing pattern in cultured erythroid cells. The level of correctly spliced transcript increased from 0.19 (day 0 after transfection) to 0.58 (day 8) in beta654/beta654 homozygous erythroid cells, and from 0.45 (day 0) to 0.83 (day 8) in beta654/betaA heterozygous erythroid cells, as determined by the ratio of normally spliced beta-globin transcript over total beta-globin transcript. Correspondingly, the ratios of globin chain biosynthesis (beta/alpha) increased from 0.16 (day 0) to 0.52 (day 8) in beta654/beta654 erythroid cells, and from 0.39 (day 0) to 0.84 (day 8) in beta654/betaA erythroid cells. Antisense RNA had no significant effect on the splicing pattern in betaA/betaA erythroid cells. The splicing pattern in transfected cells with pCMVA showed significant changes compared with that in untransfected cells and that in transfected cells with the control antisense fragment (human SRY gene sequence). In addition, we did not observe side-effects on cytological features after the introduction of pCMVA. All these results indicated that the antisense RNA transcribed from the mammalian expression vector pCMVA could efficiently and specifically suppress the aberrant splicing pattern of beta654 mutant pretranscript and restore the correct splicing pathway in vivo, leading to the improvement of globin chain biosynthesis in thalassaemic cells.

[A study of transgenic IFV cattle integrated with human serum albumin gene].

The mammary gland expression vector (pcDNA 3.1-GCALBm) containing the full-length sequence of human serum albumin (hALB) cDNA and intron 1 as well as the goat beta-casien gene promoter and 5' up-stream regulatory sequence was constructed. The vector was micro-injected into bovine IVF eggs. The embryos were in vitro cultured to the late stage of morulae, and then few embryo cells were aspirated for the implantation detection of target gene integration and SRY DNA sequence using nested-PCR. Afterwards, ten integrated embryos were selected to transfer into eight recipients and three were pregnant. The pregnant rate was 37.5%(3/8). However, two were miscarried in mid-trimester but one was pregnant at term to deliver a male transgenic cattle integrated with hALB mini-gene. The transgenic efficiency was 12.5% (1/8).

The delta-globin RNA transcript level in beta-thalassemia carriers.

Increased levels of hemoglobin A(2) (HbA(2)) are present in most beta-thalassemia carriers. The mechanism of this effect is not understood, although the increase may result from transcriptional and posttranscriptional changes. In the present study, we quantitate delta-globin mRNA levels in peripheral-blood-enriched reticulocytes and characterize the variation of delta-mRNA levels in 30 beta-thalassemia heterozygotes who individually carry one of the four common Chinese beta-thalassemia alleles [codons 41/42 (-TTCT); codon 17 (A-->T); IVS-II-654 (C-->T); -28 (A-->G)]. A sensitive and quantitative competitive reverse-transcriptase polymerase chain reaction method was developed and used to assess the absolute amounts of delta-mRNA transcripts in these peripheral erythroid cells. The results showed a large increase in delta-mRNA amounts in all the carriers examined (72.3 +/- 9.0 amol/microg RNA) as compared with those in 12 controls (1.2 +/- 0.2 amol/ microg RNA). There was a direct correlation between the delta-mRNA levels and types of beta-thalassemia alleles; generally, the delta-mRNA levels are higher in heterozygotes for beta(0)-thalassemia mutations than beta(+)-thalassemia mutations. The delta-mRNA levels correlated inversely with hemoglobin and red cell indices but directly with HbA(2) levels in heterozygotes of each of the group of beta-thalassemia mutations. These results suggest that a greater impairment in beta-globin gene expression results in increased transcription of delta-globin gene and in a higher level of HbA(2).

Molecular diagnosis of hemophilia A in Chinese patients by an analysis of inversions in the factor VIII gene.

Hemophilia A is an X-linked bleeding disorder caused by deleterious mutations in the factor VIII gene. An inversion caused by introchromosomal homologous recombination between the A gene located in intron 22 of the factor VIII gene and one of the two telomeric A genes has been recently described as the common cause of about 50% of cases of severe hemophilia A. The rearrangement can be readily detected by a Southern blotting procedure. We report use of this procedure to detect rearrangements in 106 unrelated Chinese hemophilia A cases. In 49.3% of the patients with severe disease an inversion was found, but no inversion was detected in any of the patients with moderate or mild disease. The majority of inversions (91.4%) involved the most distal A gene; in a minority (8.6%) the more proximal A gene was involved. These results indicate that intron 22 inversion is the most important molecular defect causing Chinese hemophilia A and that analysis for intron 22 inversion may be the first-line test in the molecular diagnosis of severe hemophilia A.

Prenatal Diagnosis of ß-Thalassemia Using Multiplex Allele-Specific Amplification (MASPCR) in Chinese Families.

Combining allele-specific amplification (ASPCR) with multiplex PCR, we developed a new approach-multiplex allele-specific amplification (MASPCR) to detect five common types of ß-thalassemia mutations (CD 41-42(-4bp), CD 17 A→T, CD 71-72 (+ A), -28 A→ G and IVS-2-654 C→ T) which account for approximately 80% of all ß-thalassemia alleles in Chinese individuals. Using this technique, prenatal diagnoses were performed for ten pregnancies at risk for ß-thalassemia. All the results were confirmed by PCR/ASO probe hybridization or DNA sequencing. This study suggests that this method is a simple, accurate approach that may further improve the prevention programs for ß-thalassemia that have already dramatically lowered the birth rate of affected children in some parts of the world.

Hydroxyurea therapy in beta-thalassaemia intermedia: improvement in haematological parameters due to enhanced beta-globin synthesis.

The beta-thalassaemias represent a heterogenous group of diseases resulting from decreased erythroid beta-globin mRNA expression and imbalanced alpha/beta-globin chain synthesis which are manifest clinically by ineffective erythropoiesis and excessive haemolysis. Increasing levels of haemoglobin F (HbF) by pharmacological agents has been proposed to ameliorate the severity of the disease by improving the balance in globin chain synthesis. Hydroxyurea (HU), as an effective agent with low toxicity for activating gamma-globin gene, has been shown to enhance HbF synthesis in experimental animals and in patients with sickle cell anaemia. However, previous trials of HU in beta-thalassaemia patients are ambiguous, with a small number having increased HbF synthesis. In a recent study of HU effects in Chinese beta-thalassaemia patients we unexpectedly found that two unrelated patients with beta-thalassaemia intermedia demonstrated an improvement in the effectiveness of erythropoiesis reflected by an increase in haemoglobin concentration (from 4.1 to 6.3 g/dl, patient 1; from 6.5 to 9.7 g/dl, patient 2) and in red cell volume (from 68 to 104 fl, patient 1; from 68 to 85 fl, patient 2) after a period of excess of 300d of low-dosage HU treatment. These effects, however, appear to be due to increased beta-globin biosynthesis, because the percentage of HbF decreased in each patient as total Hb increased. This was reflected by changes in the beta/alpha ratio (from 0.301 to 0.581, patient 1; from 0.348 to 0.487, patient 2) with minimal changes in gamma-globin biosynthesis. We conclude that in addition to its known effects in stimulating gamma-globin production, hydroxyurea may have a more general role in augmenting globin synthesis, including beta-globin in some thalassaemia intermedia patients who maintain the capacity to express normal beta-globin chains.

RNA transcripts of the beta-thalassaemia allele IVS-2-654 C-->T: a small amount of normally processed beta-globin mRNA is still produced from the mutant gene.

IVS-2-654 C-->T is a common Chinese beta-thalassaemia mutation. Previous studies report that this mutation resulted in the formation of an abnormally spliced mRNA and the absence of detectable normal beta-globin mRNA, hence the mutation was considered to cause beta o-thalassaemia. We recently used the method of PCR amplified cDNA copies of circulating erythroid cell mRNA to analyse the mutant gene transcripts and found that this IVS-2-654 mutation does not abolish normal RNA processing entirely, but that a significant amount (over 15%) of normally processed beta-globin mRNA is produced. Microglobin chain biosynthetic analysis using the HPLC method showed that beta-globin chain was also present in the blood of patients with IVS-2-654 C-->T mutation. Accordingly, this mutant allele leads to a beta (+)-thalassaemia. Further, the methodology described in this paper provides a new approach towards the detection of RNA transcripts of beta-thalassaemia alleles as well as the study of gene expression in beta-thalassaemia and other genetic diseases.

Treatment of beta-thalassemia with hydroxyurea (HU)--effects of HU on globin gene expression.

A newly developed method of RT-PCR/competitive PCR for measuring the relative and absolute content of globin mRNAs as well as micro-globin chain biosynthetic assay have been used to study the alterations of globin gene expressions in the patients with beta-thalassemia pre- and post-hydroxyurea (HU) treatment. It was found for the first time that HU had the effect of enhancing beta-globin gene expression in some patients. Two cases with beta-thalassemia who were subjected to HU treatment for over two years showed a marked increase in beta-globin mRNA level and beta-globin chain synthesis, resulting in more effective erythropoiesis and the alleviation of clinical symptoms.

Sexing bovine embryos using PCR amplification of bovine SRY sequence.

This study analyses the bovine SRY DNA sequence by direct sequencing procedure, followed by the designation of the PCR primers specific for bovine SRY. Using PCR amplification of bovine SRY gene, the embryo sex was determined. The results of the embryo sex identification were confirmed after the embryo transfer and pregnancies.

A new de novo mutation (A113T) in HMG box of the SRY gene leads to XY gonadal dysgenesis.

We describe a new point mutation in the SRY gene of a Chinese XY female with gonadal dysgenesis (Swyer syndrome). Using the double stranded DNA cycle sequencing method, a single nucleotide substitution of G-->A was identified at codon 113 of the patient's SRY gene, resulting in a conservative amino acid change from alanine (A) to threonine (T) at a residue that lies within the putative DNA binding motif. With this mutation, one MnlI recognition site is abolished and a new BsmAI site is present in the DNA sequence of the SRY gene; therefore, it is easily detected by analysis of the digestion of the amplified SRY DNA fragment on an electrophoretic agarose gel. In situ hybridisation to the XY female's chromosomes showed that her mutant SRY gene was indeed located on the short arm of her Y chromosome. The SRY mutation in the XY female reported here occurred de novo, as sequence analysis showed that it was not present in her father or other family members.

Study of the RNA splicing defect in the common Chinese beta-thalassemia gene, IVS-II nt. 654 C-->T by using mRNA/PCR.

With direct sequencing of the amplified cDNA, we analysed the transcript and mRNA splicing defect in a common Chinese beta-thalassemia mutant (IVS-II nt. 654 C-->T). The result shows that this mutant gene would not only produce abnormally processed beta-globin mRNA, but also transcribes a small amount of normally spliced mRNA, hence leading to beta+ thalassemia. The method described herein provides a simple and sensitive approach to the studies of gene expression and molecular defects in genetic diseases at transcriptional level.

Detection of molecular deletions in the Chinese DMD patients using two amplified dystrophin sequences.

This Brief Communication reports the detection of molecular deletions in Chinese DMD patients using two new amplified dystrophin DNAs involving the regions of exon 49 and 50. The results show that over 50% of the DMD deletions can be rapidly detected by PCR amplification of these two dystrophin sequences.

A new silent mutation found in the Chinese PAH locus and its role in the prenatal diagnosis of phenylketonuria.

A silent mutation or sequence polymorphism. A to T substitution at codon 399 in exon 11 of the PAH gene from a Chinese PKU patient, was found by sequence analysis. The frequencies of this new mutation in normal and abnormal (PKU) genes were 0.005 and 0.09, respectively, based on the analyses of 100 normal individuals and 39 PKU patients using DNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridization methods. This silent mutation can be used as a "genetic marker" for PKU prenatal diagnosis. Recently, a fetus at risk for PKU, who could not be completely predicted by RFLPs linkage analysis, was prenatally diagnosed with this genetic marker.

Analysis of RFLPs and DNA deletions in the Chinese Duchenne muscular dystrophy gene.

Sixty-nine unrelated Chinese DMD patients were studied with a series of genomic and cDNA probes. Analysis of 13 polymorphic sites showed that pERT87-1, 87-8, 87-15, and XJ probes gave favourable allele frequencies in the Chinese population, and nearly 90% of the DMD families in this study were informative for prenatal diagnosis and carrier detection using these four polymorphic markers. Nine out of 69 (13%) were also found to have gene deletions using a panel of genomic probes. However, when using cDNA probes, deletions were found in 56.5% of the patients. The deletions were concentrated in the areas of probes 7 and 8, giving a proportion of about 80% of all deleted patients in this study. All these results provide valuable information for planning prenatal diagnosis programmes for DMD in China.

PAH 399 GTA (Val)----GTT(Val), a new silent mutation found in the Chinese.

A silent mutation or sequence polymorphism, an A to T substitution at codon 399 in exon 11 of the phenylalanine hydroxylase (PAH) gene has been identified by DNA sequence analysis in the Chinese. The frequencies of this new mutation in normal and abnormal (phenylketonuria: PKU) genes are 0.005 and 0.09, respectively, based on the analyses of 100 apparently normal individuals and 39 PKU patients, as demonstrated by DNA amplification with polymerase chain reaction (PCR) and oligonucleotide hybridization methods. The results suggest that there is linkage disequilibrium between this polymorphism and PKU mutations in the PAH gene; approximately 10% of defect PAH alleles in the Chinese population may be identified with this sequence polymorphic marker.

Prenatal detection of an Arg----Ter mutation at codon 111 of the PAH gene using DNA amplification.

A CGA----TGA mutation at codon 111 in exon 3 of the phenylalanine hydroxylase (PAH) gene was recently identified in a Chinese phenylketonuria (PKU) patient. This paper reports the prenatal diagnosis of a Chinese fetus at risk for PKU using DNA amplification with PCR and oligonucleotide hybridization. RFLP analysis revealed that the fetus had inherited a PKU gene from his mother, but his paternal PAH gene was uninformative. PCR amplification of 300 bp which included exon 3 plus the flanking intronic sequences of the PAH gene was performed. The amplified DNA was hybridized with a pair of allele-specific oligonucleotide probes. The results indicated that the fetal DNA carried a PAH 111 Arg----Ter mutant gene inherited from his father. Thus, the fetus was predicted to be affected with PKU.