Cloning and sequencing of buffalo male-specific repetitive DNA: sexing of in-vitro developed buffalo embryos using multiplex and nested polymerase chain reaction

Buffalo Y-chromosome specific repetitive DNA (BuRY.I) was cloned and sequenced in order to develop a sensitive method for sexing of buffalo preimplantation stage embryos using polymerase chain reaction (PCR). A highly sensitive and reliable sex determination assay using a primary (BRY.I), nested (BuRYN.I) and multiplex (BuRYN.I, ZFX/ZFY) PCR was developed. The BRY.I and BuRYN.I primers are targeted to amplify Y-specific sequences, while the ZFX/ZFY loci was amplified to serve as a positive control for both male and female samples. Accuracy of the sex determination assay was initially verified with genomic DNA obtained from blood of known gender. Further sensitivity and reproducibility of the assay was examined using DNA obtained from 1 or 2 blastomeres to demi embryos. Altogether, 80 IVF-derived embryos ranging from the 2 to 4 cell to the blastocyst stage were used for sex determination. Definite and clear signals following PCR amplification were obtained from all embryo samples. Accuracy of assays was determined by comparing results from a single cell with those of blastocyst stage embryos, thereby indicating that 1 or 2 blastomeres from a preimplantation buffalo embryo is sufficient for sex determination by PCR. No misidentification was observed within the embryo samples using nested (BuRY.I), primary (BRY.I) and multiplex (BuRYN.I; ZFX/ZFY) PCR, suggesting that this technique is a highly reliable method for sexing buffalo embryos.     

Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.     

Amplification and application of the HMG box of bovine SRY gene for sex determination

A fast and reliable method for bovine sexing has been developed through amplification of the bovine high motility group (HMG) box of the sex-determining region of the Y chromosome gene (SRY). Oligonucleotide primers were designed according to the conserved bovine SRY HMG box sequence motif. In agarose gel electrophoresis, a normal bull showed 1 SRY band, and a normal cow showed no SRY band. After optimization, the PCR procedure for sex determination was applied to 14 embryo biopsies. The biopsied embryos were transferred into 14 recipient cows on the same day (day 7 of the estrus cycle) that the embryos were collected and sex of the calf was confirmed after parturition. Nine calves were born and anatomical sex corresponded to those sex determined by PCR in all cases (100% accuracy). Thus, this study showed for the first time that the present method can be applied in bovine breeding programs to facilitate manipulation of the sex ratio of offspring and also allows a quick diagnosis for the XY-bovine offspring by amplification of the HMG box of the bovine SRY gene.     

小麂Sry基因的克隆和测序

应用人的性别决定基因SRY(Sex-determining Region Y gene,SRY)中HMG框内的一对引物,对小麂细胞株的基因组DNA进行PCR扩增,得到雄性小麂细胞的220bp扩增产物,而在雌性小麂细胞中未发现扩增产物。将雄性小麂细胞的220bp扩增产物通过T-A互补法克隆到质粒pGEM-T 载体中,筛选阳性克隆进行DNA测序。测序结果表明小麂Sry基因保守序列与人的SRY基因保守区相同碱基的比值为152/184,达到82.6%。提示小麂Sry基因与人的SRY基因存在着较高的同源性,说明SRY基因在进化过程中高度保守。 Abstract:Using the primers from SRY gene——HMG Box for PCR amplification in genomic DNA of Muntiacus reevesi cell strains,a 220bp fragment was obtained in the male but not in the female.The 220bp fragment was cloned into the pGEM-T vector using T/A clone method.The identified positive clone was sequenced.The result shows that 82.6% nucleotides(152bp/184bp) are homologous between Muntiacus Sry and human SRY gene.It suggests that SRY is highly conserved during evolution.     

Sex determination of in vitro developed buffalo (Bubalus bubalis) embryos by DNA amplification

This study was conducted to determine the sex of buffalo embryos produced in vitro by amplifying male specific DNA sequences using the polymerase chain reaction (PCR). This method uses three different pairs of bovine Y-chromosome specific primers and a pair of bovine satellite specific primers. Buffalo in vitro fertilized embryos at the 4-cell to blastocyst stage were collected at days 3, 4, 6, and 8 postinsemination, and the sex of each embryo was determined using all three different Y-chromosome specific primers. The bovine satellite sequence specific primers recognize similar sequences in buffalo and are amplified both in males and in females. Similarly, Y-chromosome specific primers amplify the similar Y-chromosome specific sequences in male embryos of buffalo. Upon examining genomic DNA from lymphocytes of adult males and females, and embryos, the results demonstrate the feasibility of embryo sexing in buffaloes. Furthermore, sex determination by PCR was found to be a rapid and accurate method. © 1993 Wiley-Liss, Inc.     

Fast and reliable sexing of prosimian and human DNA

Molecular sexing of mammals is normally done by PCR amplification of Y chromosomal fragments, or coamplification of homologous fragments from both sex chromosomes. Existing primers are often unreliable for distantly related species due to mutations in primer regions. Currently there are no published primers for the sexing of prosimian DNA. We show that an existing method (using the zinc finger protein) based on a size difference between the X and Y homologs does not work in prosimians. Multiple alignments of distantly related mammalian species from Genbank and genome databases enabled us to identify conserved regions in the amelogenin gene. Using these conserved regions, we can target species that have no sequence information. We designed a single, conserved primer pair that is useful for fast and reliable molecular sexing of prosimian primates. A single PCR yields two fragments in males and only one in females, which are easily separated with the use of agarose gels. Amplification of separable fragments was successful in seven species of lemurs, as well as humans.     

Gender determination in single bovine blastomeres by polymerase chain reaction amplification of sex-specific polymorphic fragments in the amelogenin gene.

A sensitive technique for the sexing of bovine embryos was developed using polymerase chain reaction (PCR) amplification of the bovine amelogenin (bAML) gene on the X- and Y-chromosomes of Holstein dairy cattle. Cloning and DNA sequencing showed a 45.1% homology between the fifth intron of the bAML-X and bAML-Y gene with multiple deletions. A pair of sex-specific primers was designed to allow amplification of a single fragment of 467-bp from the X-chromosome of female cattle and two fragments of 467-bp and 341-bp from the X- and Y-chromosomes of male cattle. The primers were successfully applied to bovine sexing from single blastomeres isolated from day-6 to day-7 cow embryos by direct cell lysis and PCR. Our protocol of embryo sexing should be applicable to the diagnosis of defective genes in vitro in human embryos and in other domestic or recreational animals.     

Determination of sex and chimaerism in the domestic sheep by DNA amplification using HMG-box and microsatellite sequences

We describe a rapid, reliable method for the sexing of the domestic sheep (Ovis aries) by amplification of Y-chromosome-specific sequences in male genomic DNA using the polymerase-chain reaction (PCR). Oligonucleotide primers were selected from a conserved sequence, the HMG box, in the sequence of ovine Sry, permitting amplification of a defined 161 bp fragment only from male-specific genomic DNA. As a control, microsatellite primers also were used in PCR reactions, recognising a sequence that is amplifiable in genomic DNA from both males and females. In addition, we demonstrate the feasibility of using this technique for the detection of Y-specific sequences in foetal biopsies (specifically small numbers of foetal germ cells), and in reconstruction mixtures of male and female genomic DNA to simulate the analysis of intersex chimaeras which would be produced when pluripotent cells have been established for this species.     

Sex determination in goat by amplification of the HMG box using duplex PCR

The objective of this study was to obtain a fast, accurate and reliable method of determining the sex of goat embryos prior to implantation through amplification of the high-motility-group (HMG) box of the sex-determining region of the Y chromosome (SRY) gene of the goats. Goat specific primers were designed for duplex polymerase chain reaction (PCR). As an internal control gene, the goat beta-action gene sequence was simultaneously amplified together with the HMG box of goat SRY gene. Males showed both 1 SRY band and 1 beta-action band, but only 1 beta-action band was present in the agarose gel electrophoresis of females. The result indicated that the goat HMG-box sequence motif of SRY was male specific. Afterward, the optimized PCR procedure was applied in 30 embryo biopsies and the biopsied embryos were transferred into 30 recipient female goats. The sex of the 13 kids proved anatomically corresponded to the sex determined by PCR (100% accuracy). Thus, this study showed that this duplex PCR method can be applied to sex the goat pre-implantation embryos and to manipulate the sex ratio of offspring in goat breeding programs.     

A rapid improved method for sexing embryo of water buffalo

The objective of the experiment of this paper is to develop and improve in the sexing method for preimplantation embryos of water buffalo (Bubalus bubalis) using loop-mediated isothermal amplification (LAMP) reaction. Embryo sexing has been recognized to control effectively the sex of offspring in the embryo transfer industry. A rapid and simple detection system was established by adding ethidium bromide (EB) or 5μl of CuSO4 (3M) to the product of LAMP reaction. The result of these additions after 2 min was a color change and a precipitate. It could be employed as an alternative method in the detection of the reaction products in place of the time consuming electrophoresis or the turbidity meter. The in vitro produced buffalo embryos were divided into one to eight pieces using a microblade attached to a micromanipulator. The cell number in each piece was counted before sexing. Sexing of DNA samples extracted from one to five biopsies cells was performed by LAMP. After biopsy, the remaining part of the embryos was used to confirm the sex by polymerase chain reaction (PCR). Fifty buffalo embryos were used and the accuracy of sex prediction was 100% when the blastomeres dissociated from a morula exceeds three. In conclusion, the present procedure without turbidity meter and electrophoresis was reliable and applicable for sexing the water buffalo embryos.     

PCR扩增泥鳅和大鳞副泥鳅SRY盒基因

以特异扩增人ＳＲＹ基因保守区的一对引物,研究了泥鳅和大鳞副泥鳅基因组中ＳＲＹ盒基因的扩增。结果表明,该引物可以在泥鳅中扩增出四条带,其长度分别为２００,５５０、９４０和１０００ｂｐ。在大鳞副泥鳞中扩增出三条带,大小为２００,５５０和９００ｂｐ。经Ｓｏｕｔｈｅｒｎ杂交显示出二者的阳性带为２００和５５０ｂｐ。阳性带在雌雄个体间和两个物种间无差异。     

Sex diagnosis of equine preimplantation embryos using the polymerase chain reaction

A rapid and reliable method for sex determination of preimplantation-stage equine embryos has not been available. The aim of the present study was to find an enzyme which would distinguish sexes in the horse by finding a polymorphic restriction site between the ZFY and ZFX homologues amplified by the polymerase chain reaction (PCR). Altogether, 38 different restriction enzymes were tested using female and male DNA extracted from blood. The primers used for amplification were selected from conserved sequences between human ZFY and ZFX genes and mouse Zfy-1 and Zfy-2 genes. Nine enzymes cut the PCR product of approximately 450 basepairs, but only Bsm I yielded different banding patterns in female and male DNA. All blood samples were correctly diagnosed. To test the method on embryonic cells, 17 horse demi-embryos were obtained from expanding blastocysts 220 to 950 mum in diameter. Demi-embryos were further cut into 3 to 7 parallel samples which were analyzed individually to test the repeatability of the method. Eight of the original embryos were diagnosed as females and 9 as males. No misidentifications were observed within the embryonic samples, suggesting that this sexing method is highly reliable. This study provides a rapid and accurate method to sex horse embryos.     

水牛SRY基因的克隆及序列分析

根据荷斯坦牛SRY基因设计一对引物,采用聚合酶链式反应（PCR）技术,以中国沼泽型水牛（Swamp Buffalo）基因组DNA为模板,扩增得到SRY（Sex Deterimation region of Y chromosome）全序列约2005bp,其中1-504bp为5’启动子区,1196-2005bp为3’侧翼序列,在505-1195bp为SRY的外显子,编码229个氨基酸。在SRY HMG box区域设计探针,用地高辛标记后分别与雄性、雌性水牛基因组DNA进行Southern 杂交,结果显示该段序列只在雄性DNA样本中有杂交信号,证明SRY基因为雄性特异。BLAST比对结果显示与牛属动物SRY基因的同源性为96％,其中SRY基因HMG box区域同源性高达99%,说明SRY基因具有高度的进化保守性     

Cloning of Taiwan water buffalo male-specific DNA sequence for sexing

Random amplified polymorphic DNA (RAPD) fingerprinting was carried out to investigate the sex-specific DNA sequence for sexing in Taiwan water buffalos. One hundred and forty random primers were used for RAPD-PCR (polymerase chain reaction). One of these primers, OPC-16, produced a 321 bp fragment found only in tested males. This male-specific fragment was isolated and constructed into plasmids for nucleotide sequencing, a novel male-specific sequence was obtained. Two primers (BuSexOPC16-F and -R) were designed according to the cloned male-specific sequence to amplify the male-specific fragment using PCR for sexing. Sex-specific bands in the gel were represented in the males but none were found in the females when the Taiwan water buffalo genomic DNA samples were amplified with these two primers using PCR. The same results were also obtained from Taiwan yellow, Holstein, Angus, and Hereford cattle samples. This showed that the sex of these five breeds could be easily and effectively determined using the PCR technique.     

一例性反转畸形SRY基因片段的扩增、克隆和核苷酸序列分析

在哺乳动物中,位于Ｙ染色体上的指导雄性性别分化的基因被命名为睾丸决定因子（Ｔｅｓｔｉｓ－ｄｅｔｅｒｍｉｎｉｎｇｆａｃｔｏｒ,ＴＤＦ）１９９０年６月分离获得的ＳＲＹ基因（Ｓｅｘ－ｄｅｔｅｒｍｉｎｉｎｇｒｅｇｉｏｎｏｆｔｈｅＹ）被认为是ＴＤＦ基因最好的候选者［１－４］。ＳＲＹ基因为单拷贝,位于Ｙ染色体短臂末端１Ａ１Ａ区,靠近假常染色体配对区（ＰＡＰＲ）的交界处,其部分顺序编码８０个保守性氨基酸组成的多肽。本实验使用与ＳＲＹ基因相应的引物,利用ＰＣＲ技术以一例性反转畸形病人的基因组ＤＮＡ为模板分离ＳＲＹ基因保守区顺序,并将特异扩增出的此ＳＲＹ基因片段重组到质粒ｐＵＣ１２中,得到含有ＳＲＹ基因片段的克隆。经测序表明其ＳＲＹ基因保守顺序上有Ｔ→Ｃ（Ｓｅｒ→Ｐｒｏ）突变。ＳＲＹ基因的存在及其突变可能是导致性反转畸形发病的原因。     

Ovine-specific Y-chromosome RAPD–SCAR marker for embryo sexing

An accurate, sensitive, and quick (approximately 3 h) method for determining the sex of ovine embryos was developed using polymerase chain reaction (PCR) primers derived from an ovine-specific Y-chromosome random amplified polymorphic DNA marker ( UcdO43 ). The accuracy and sensitivity of the assay were first tested using genomic DNA from 10 males and 10 females of five different sheep breeds, and then tested using serial dilutions of male-in-female DNA. The assay was 100% accurate in confirming the sex of the individuals and the ovine male-specific fragment was detected in dilutions containing as little as 10 pg of male DNA in 50 ng of female DNA. The assay was also confirmed to be specific for the ovine Y-chromosome as bovine, caprine, porcine, murine, and human DNA did not amplify. The ovine embryo sexing method is a duplex PCR system that also includes ZFY/ZFX primers. ZFY/ZFX provide an internal positive control for amplification as well as a means to confirm the results obtained with the UcdO43 primers. All embryo sexing results (36/36) from our method were in agreement with the ZFY/ZFX assay results. However, while our method requires an internal control to detect PCR failure, it has the advantages of not requiring nested PCR or restriction endonuclease digestion of the PCR product, and concerns about cross-species contamination are eliminated.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60 degrees C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Preliminary development of a diagnostic test for Brucella using polymerase chain reaction

A highly sensitive and specific diagnostic test for Brucella based on polymerase chain reaction is under development in our laboratory. A commercially available PCR kit was used to create primers that allowed the amplification of a 635 bp fragment of a 43 kDa outer membrane protein gene from Brucella abortus strain 19. We successfully amplified the cloned gene present in the pMS64 plasmid and genomic Brucella S19 DNA. The amplified DNA was easily detected by agarose gel electrophoresis. Using both the pMS64 plasmid and Br. abortus S19 purified DNA as template each component of the PCR reaction was adjusted for the optimum amplification of the DNA sequence. Optimum specific amplification resulted when the primer annealing temperature was 60C. The gene fragment was amplifiable in 25 different Brucella species and strains. To test the specificity of the reaction, DNA extracted from 17 micro-organisms possibly associated with cattle were tested. No amplification was observed. The sensitivity of the reaction was determined with different concentrations of genomic Brucella strain 19 DNA. As little as 0.1 pg DNA (less than 100 brucella cells) could be detected. The specificity and sensitivity of PCR combined with its simplicity and speed suggests the potential of this technique for routine diagnosis of brucellosis.     

Rapid sexing of murine preimplantation embryos using a nested,multiplex polymerase chain reaction (PCR)

The objective of this study was to develop a rapid and efficient means of sexing murine preimplantation embryos at the 4- to 8-cell stage of development. To achieve this goal, a nested, multiplex polymerase chain reaction (PCR) was optimized using DNA from male and female mice and primers specific for X- (DXNds3)- and Y- (Sry,Zfy) gene sequences. Sensitivity of the assay was measured using groups of 4, 2, or 1 blastomere from dissociated embryos. Efficiency was evaluated using single blastomeres obtained by embryo biopsy. Accuracy of sexing was determined by comparing single-cell results with those of matched blastocysts. Robust amplification of male (XY) and female (XX) gene sequences was obtained in less than 6 hours. The percentage of male (3 bands) and female (1 band) reactions for groups of 4, 2, or 1 blastomere was 100% (6/6), 100% (15/15), and 94.4% (17/18), respectively. Assay efficiency for single, biopsied blastomeres from 4 to 8 cell embryos was 95.8% (207/216). For male and female embryos, sexing of single blastomeres accurately predicted results of matched blastocysts, 100% (10/10) and 100% (13/13), respectively. Simultaneous amplification of one X- and two Y-gene sequences ensured correct interpretation of sexing reactions. Short thermal cycling times and minimal tube handling increased the assay speed and decreased the potential risk of contamination. Mol. Reprod. Dev. 49:261–267, 1998. © 1998 Wiley-Liss, Inc.     

Sexing and detection of gene construct in microinjected bovine blastocysts using the polymerase chain reaction

We present a polymerase chain reaction (PCR)-based procedure for rapid bovine embryo sexing and classifying embryos for the presence of exogenous DNA. Fourteen bovine blastocysts microinjected with gene construct DNA at the pronuclear stage were divided into quarters and subjected to amplification with construct-specific and sex gene-specific (ZFY/ZFX) primers in the same initial PCR reaction. Blastocysts carrying microinjected construct DNA could be identified by the presence of construct-specific PCR product in approximately 4 h. Approximately half of the microinjected and two of 16 non-microinjected blastocysts typed PCR-positive for the construct DNA. Owing to erroneous amplifications in the two non-microinjected control blastocysts, and the inability of the system to distinguish integrated from non-integrated copies of the microinjected construct, the number of construct-positive blastocysts determined in our assay most likely overestimates the number of true transgenic embryos. Nevertheless, using this assay, we were able to determine that approximately half of the microinjected embryos were negative for the transgene construct and thus could be eliminated from transfer to a recipient cow. Embryo sexing was achieved in less than 6 h by restriction fragment length polymorphism analysis of nestedZFY/ZFXPCR products reamplified from initial PCR reactions. In 11/14 microinjected blastocysts all sections assayed unambiguously as the same sex. In one embryo, only one section was analysed, while two other blastocysts whowed some discrepancies of sexing results between the sections analysed. The approach employed here to determine the sex and presence of microinjected construct DNA in bovine preimplantation embryos is rapid, accurate among different sections of an embryo and can be used to increase the efficiency of current transgenic cattle production procedures.