中国南方低丘红壤区人工复合生态系统模式建造研究

中国南方低丘红壤区人工复合生态系统模式建造研究徐盛荣,吴珊眉,李辉信（南京农业大学土壤农业化学系,２１００１４）ＭｏｄｅｌｉｎｇｏｆＡｒｔｉｆｉｃｉａｌＣｏｍｐｌｅｘＥｃｏｓｙｓｔｅｍｉｎＬｏｗ－ｈｉｌｌｙＲｅｄＳｏｉｌＲｅｇｉｏｎｏｆＳｏｕｔｈＣｂｉｎａ￥．ＸｕＳｈｅｎｇｒｏｎｇ;ＷｕＳｈａｎｍｅｉ;ＬｉＨｕｉｘｉｎ（ＤｅｐａｒｔｍｅｎｔｏｆＳｏｉｌＳｃｉｅｎｃｅａｎｄＡｇｒｉｃｕｌｔｕｒａｌＣｈｅｍｉｓｔｒｙ,ＮａｎｊｉｎｇＡｇｒｉｃｕｌｔｕｒａｌＵｎｉｖｅｒｓｉｔｙ）．ＣｈｉｎｅｓｅＪｏｕｒｎａｌｏｆＥｃｏｌｏｇｙ,１９９３,１２（２）：３９—４０．Ｔｈｉｓｐａｐｅｒｒｅｓｅａｒｃｈｅｓｉｎｔｏｔｈｅｍｏｄｅｌｉｎｇｏｆａｒｔｉｆｉｃｉａｌｃｏｍｐｌｅｘｅｃｏｓｙｓｔｅｍｉｎｃｏｒｐｏｒａｔｉｎｇｃｒｏｐ,ｆｒｕｉｔ,ｆｏｒｅｓｔａｎｄａｎｉｍａｌｈｕｓｂａｎｄｒｙｗｉｔｈｉｎａｃａｔｃｈｍｅｎｔａｒｅａ,ｗｈｉｃｈｉｓａｂａｓｉｃｕｎｉｔｏｆｌｏｗｈｉｌｌｙｒｅｇｉｏｎｉｎｓｏｕｔｈＣｈｉｎａ．Ｔｈｅｍｏｄｅｌｐｒｏｄｕｃｅｓｓｉｇｎｉｆｉｃａｎｔａｎｄｃｏｍｐｒｅｈｅｎｓｉｖｅｐｒｏｆｉｔｓ．Ｆｉ     

民和盆地早白晋世晚期的孢粉组合

民和盆地的甘肃兰州柴家台地区,下白垩统河口组上亚组含有丰富的孢粉化石。上亚组上部是以Ｓｃｈｉｚａｅｏｉｓｐｏｒｉｔｅｓ－Ｃｉｃａｔｒｉｃｏｓｉｓ ｐｏｒｉｔｅｓ为代表的粉组合,并可划分出上下两个亚组合,分别以Ｃｌａｓｓｏｐｏｌｌｉｓ－Ｓｃｈｉｚａｅｏｉｓｐｏｒｉｔｅｓ和Ｐｉｃｅａｐｏｌｌｅｎｉｔｅｓ－Ｃｉｃａｔｒｉｃｏｓｉｓｐｏｒｉｔｅｓ来表示。     

伞形科植物上的柄锈菌属新种

本文报告了伞形科植物上三个柄锈菌属新种,它们是辽藁本Ｌｉｇｕｓｔｉｃｕｍ ｊｅｈｏｌｅｎｓｅ Ｎａｋａｉ＆Ｋｉｔａｇａｗａ上的辽藁本柄锈菌Ｐｕｃｃｄｉｎｉａ ｌｉｇｕｓｔｉｃｉ－ｊｅｈｏｌｅｎｓｉｓ Ｊ．Ｙ．Ｚｈｕａｎｇ＆Ｓ．Ｘ．Ｗｅｉ,波棱滇芎Ｐｈｙｓｏｓｐｅｒｍｉｐｓｉｓ ｏｂｔｕｓｉｕｓｃｕｌａ（Ｃ．Ｂ．Ｃｌａｒｋｅ）Ｎｉｒｍａｎ上的滇芎柄锈菌Ｐｕｃｃｉｎｉａ ｐｈｙｓｏｓｐｅｒｍｏｐｓｉｓ     

中国兰科植物新资料

本文报道了２个新种（元阳石豆兰ＢｕｌｂｏｐｈｙｌｌｕｍｙｕａｎｙａｎｇｅｎｓｅＴｓｕｉ,长帽隔距兰ＣｌｅｉｓｏｓｔｏｍａｌｏｎｇｉｏｐｅｒｃｕｌａｔｕｍＴｓｉ）、２个新组合（翼萼卷瓣兰ＢｕｌｂｏｐｈｙｌｌｕｍｒｅｔｕｓｉｕｓｃｕｌｕｍＲｃｈｂ．ｆ．ｖａｒ．ｏｒｅｏｇｅｎｅｓ（Ｗ．Ｗ．Ｓｍｉｔｈ）Ｔｓｉ,角萼卷瓣兰Ｂ．ｒｅｔｕｓｉｕｓｃｕｌｕｍＲｃｈｂ．ｆ．ｖａｒ．ｔｉｇｒｉｄｕｍ（Ｈａｎｃｅ）Ｔｓｉ）和１个新命名（细茎毛兰ＥｒｉａｔｅｎｕｉｃａｕｌｉｓＳ．Ｃ．ＣｈｅｎｅｔＴｓｉ）。     

苔草属植物柄锈菌国产种的补充记载

苔草属植物柄锈菌３个新种和２个中国新记录种,它们是：寄生在点叶苔草ＣａｒｅｘｈａｎｃｏｃｋｉａｎａＭａｘｉｍ．上的点叶苔草柄锈菌（新种）Ｐｕｃｃｉｎｉａｃａｒｉｃｉｓ－ｈａｎｃｏｃｋｉａｎａｅＪ．Ｙ．Ｚｈａｎｇ＆Ｓ．Ｘ．Ｗｅｉｓｐ．ｎｏｖ,寄生在十字苔草ＣａｒｅｘｃｒｕｃｉａｔａＷａｈｌｅｎｂ．上的海南柄锈菌（新种）ＰｕｃｃｉｎｉａａｈａｉｎａｎｅｎｓｉｓＪ．Ｙ．Ｚｈｕａｎｇ＆Ｓ．ＸＷｅｉｓｐ．ｎ     

辽西走廊农业生态系统区位优势分析

辽西走廊农业生态系统区位优势分析梁文举（中国科学院沈阳应用生态研究所,１１００１５）ＬｏｃａｔｉｏｎａｌＳｕｐｅｒｉｏｒｉｔｙＡｎａｌｙｓｉｓｏｆＡｇｒｏｅｃｏｓｙｓｔｅｍｓｉｎＷｅｓｔＬｉａｏｎｉｎｇＣｏｒｒｉｄｏｒ￥ＬｉａｎｇＷｅｎｊｕ（ＩｎｓｔｉｔｕｔｅｏｆＡｐｐｌｉｅｄＥｃｏｌｏｇｙ,ＡｃａｄｅｍｉａＳｉｎｉｃａ,Ｓｈｅｎｙａｎｇ１１００１５）．ＣｈｉｎｅｓｅＪｏｕｒｎａｌｏｆＥｃｏｌｏｇｙ,１９９３,１２（２）：５９－６０．Ｂａｓｅｄｏｎｔｈｅｒｅｌａｔｉｖｅａｇｇｒｅｇａｔｉｏｎｄｅｇｒｅｅｓａｍｏｎｇｈｉｅｒａｒｃｈｉｅｓｏｆｄｉｆｆｅｒｅｎｔａｇｒｏｅｃｏｓｙｓｔｅｍｓ,ｔｈｅａｕｔｈｏｒｐｒｏ－ｐｏｓｅｄｔｈａｔｌｏｃａｔｉｏｎａｌｑｕｏｔｉｅｎｔｍｏｄｅｌｃｏｕｌｄｂｅｕｓｅｄｔｏｍｅａｓｕｒｅｔｈｅｌｏｃａｔｉｏｎａｌｓｕｐｅｒｉｏｒｉｔｙｏｆａｇｒｏｅｃｏｓｙｓｔｅｍ．Ｔｈｅｍａｉｎｐｕｒｐｏｓｅｏｆｔｈｉｓｐａｐｅｒｉｓｔｏｐｒｏｖｉｄｅａｓｃｉｅｎｔｉｆｉｃｂａｓｉｓｆｏｒｃｈｏｏｓｉｎｇａｇｒｉｃｕｌｔｕｒａｌｍａｒｋｅｔａｂｌｅｂａｓｅｓａｎｄｅｓｔａｂｌｉｓｈｉｎｇａｈｉｇ     

柑橘抗寒育种早期鉴定的一种指标

以５个抗寒性不同的柑橘品种或杂交后代：“山金柑”（ＦｏｒｔｕｎｅｌａｈｉｎｄｓｉＳｗｉｎｇｌｅ）、“伏令夏橙”（ＣｉｔｒｕｓｓｉｎｅｎｓｉｓＯｓｂ．ｃｖ．Ｖａｌｅｎｃｉａ）、“伏令夏橙”＋“宁波金柑”（Ｃ．ｓｉｎｅｎｓｉｓｃｖ．Ｖａｌｅｎｃｉａ＋Ｆ．ｃｒａｓｉｆｏｌｉａＳｗｉｎｇ．ｃｖ．Ｍｅｉｗａ）、“Ｐａｇｅ”橘柚（Ｃ．ｒｅｔｉｃｕｌａｔａＢｌａｎｃｏ×Ｃ．ｇｒａｎｄｉｓＯｓｂ．ｃｖ．Ｐａｇｅ）、“Ｍｕｒｃｏｔ”橘橙（Ｃ．ｒｅｔｉｃｕｌａｔａＢｌａｎｃｏ×Ｃ．ｓｉｎｅｎｓｉｓＯｓｂ．ｃｖ．Ｍｕｒｃｏｔ）为材料,研究了低温锻炼期间柑橘原生质体抗寒性的变化及其田间植株的冬季抗寒性,表明离体原生质体抗寒性与田间植株抗寒性之间有显著的正相关,因而前者可以作为衡量后者的指标。     

中国齿裂菌属研究Ⅲ.

报道齿裂菌属的２个新种：生于青冈Ｃｙｃｌｉｂａｌａｎｏｐｓｉｓ ｇｌａｕｃａ（Ｔｈｕｎｂ．）Ｏｅｒｓｔ．及绵石栎Ｌｉｔｈｏｃａｒｐｕｓ ｈｅｎｒｙｉ（Ｓｅｅｍ．）Ｒｅｈｄ．ｅｔ Ｗｉｌｓ．上的黄山齿裂菌Ｃｏｃｃｏｍｙｃｅｓ ｈｕａｎｇｓｈａｎｅｎｓｉｓ Ｙ．Ｒ．Ｌｉｎ＆Ｚ．Ｚ．Ｌｉ ｓｏ．ｎｏｖ．和生于青冈上的大齿裂菌Ｃ．ｍａｇｎｕｓＹ．Ｒ．Ｌｉｎ＆Ｚ．Ｚ．Ｌｉ ｓｐ．ｎｏｖ．;２个中国新记录种：     

中国尾孢菌属及其近似属的研究VI.(英文)

本文报道９种尾孢菌,其中有２个新种：蟹甲草尾孢 Ｃｅｒｃｏｓｐｏｒａ ｃａｃａｌｉａｅ Ｙ． Ｌ． Ｇｕｏ　＆ Ｙ．Ｊｉａｎｇ和菜蓟尾孢Ｃｅｒｃｏｓｐｏｒａ 　ｃｙｎａｒａｅ Ｙ． Ｌ． Ｇｕｏ　＆ Ｙ． Ｊｉａｎｇ,中国新记录种有迪氏尾孢Ｃｅｒｃｏｓｐｏｒａｄｅｍｅｔｒｉｏｎｉａｎａ　 Ｇ． Ｗｉｎｔｅｒ,田菁生尾孢 Ｃｅｒｃｏｓｐｏｒａ　 ｇｌｏｔｈｉｄｉｉｃｏｌａ 　Ｔｒａｃｙ　＆ 　Ｅａｒｌｅ,甘草尾孢 Ｃｅｒｃｏｓｐｏｒａｇｌｙｃｙｒｒｈｉｚａｅ 　（Ｓａｖｕｌｅｓｃｕ　＆　Ｓａｎｄｕ）Ｃｈｕｐｐ,野桐尾孢Ｃｅｒｃｏｓｐｏｒａ　ｍａｌｌｏｔｉ　Ｅｌｌｉｓ　＆ 　Ｅｖｓｒｈ,木薯尾孢Ｃｅｒｃｏｓｐｏｒａ ｍａｎｉｈｏｂａｅ Ｖｉｅｇａｓ,补骨脂尾孢 Ｃｅｒｃｏｓｐｏｒａ ｐｓｏｒａｌｅａｅ－ｂｉｔｕｍｉｎｏｓａｅ Ｓａｖｕｌ．＆ Ｓａｎｄｕ和香豆尾孢Ｃｅｒｃｏｓｐｏｒａ ｔｒａｖｅｒｓｉａｎａ Ｓａｃｃ。文中为新种提供了拉丁文描述并附图,研究的标本保存在中国科学院微生物研究所菌物标本馆（ＨＭＡＳ）。     

天然药物会议信息(英文)

Ｊａｎｕａｒｙ 17 2 0 ,2 0 0 1:ＩｎｄｅｐｅｎｄｅｎｔＰｈａｒｍａｃｙＣｈａｉｎＣｏｎｆｅｒｅｎｃｅ ,ＳｃｏｔｔｓｄａｌｅＰｒｉｎｃｅｓｓＲｅｓｏｒｔ,Ｓｃｏｔｔｓｄａｌｅ,Ａｒｉｚｏｎａ .Ｃｏｎｔａｃｔ:ＮＣＰＡＥｘｈｉｂｉｔｓｍａｎａｇｅｒ( 70 3 )683 3 619(ｆａｘ) .Ｆｅｂｒｕａｒｙ 2 0 2 3 ,2 0 0 1:ＥＸＰＯ 2 0 0 1,ＮａｔｉｏｎａｌＣｏｍｍｕｎｉｔｙＰｈａｒｍａｃｉｓｔｓＡｓｓｏｃｉ ａｔｉｏｎ ,ＣｈａｉｎＤｒｕｇＭａｒｋｅｔｉｎｇＡｓｓｏｃｉａｔｉｏｎ ,ＮａｔｉｏｎａｌＨｏｍｅＩｎｆｕｓｉｏ…     

四倍体补偿技术的建立及其应用

常规基因剔除小鼠的获得主要是利用ES细胞的全能性先获得嵌合体小鼠,再利用:ES细胞的生殖系传递能力,通过嵌合体与野生型小鼠的交配获得杂合子小鼠.而四倍体补偿技术则可绕过嵌合体小鼠阶段,直接获得基因修饰杂合子小鼠.利用电融合技术和Piezoelectric microinjecfion显微注射技术建立了四倍体补偿技术,小鼠四倍体胚胎的获得率(电融合率)为(93.01±l.37)%,经体外培养囊胚形成率为(82.49±2.08)%.通过显微注射方法将2种129品系小鼠来源的ES细胞(CJ7和SCR012)注射到四倍体囊胚腔中,获得了完全ES细胞来源的小鼠,ES鼠的获得率分别为2.7%和8.3%.经微卫星DNA检测,成体小鼠的10个被检测组织均为129小鼠来源的.同时,也利用基因修饰的ES细胞进行了研究,获得了2种基因修饰的完全ES细胞来源的杂合子小鼠,部分小鼠具有繁殖能力,经繁育已获得了纯合子,其中凝血因子Ⅷ基因敲除小鼠获得了预期的血友病小鼠表型.上述结果说明四倍体补偿技术可应用于基因修饰小鼠的制备.     

ES细胞体外定向分化为成熟肝细胞的实验研究

探讨了肝细胞在胚胎干细胞（ES cell）体外诱导分化系统中成熟分化的条件、机制及其鉴定方法.利用TGF, bFGF、HGF等细胞生长因子进行BALB/c小鼠ES细胞向肝细胞方向的定向诱导.利用反转录聚合酶链反应（RT-PCR）、免疫细胞化学（ICC）和放射免疫法（RIA）动态检测肝细胞特异性基因和蛋白AFP,ALB,G6P,TAT,CK8, CK18等在培养体系中的表达,并测定肝细胞的尿素合成功能,最后测定肝细胞分化率.结果,肝细胞特异基因AFP, ALB,G6P和TAT最早分别于第3、9、11、13天表达,肝细胞特异蛋白AFP,CK8,CK18和ALB最早分别于第7、9、9和11天开始表达.第12天开始检测到尿素出现,浓度为8.3 μmol/L,并随培养时间延长而浓度逐渐增加.最后,测得生长因子诱导组肝细胞的分化率为32％,对照组肝细胞分化率为8%.说明肝细胞可以在ES细胞体外诱导分化系统中出现并成熟分化,bFGF、HGF、OSM等可以明显提高细胞分化率和成熟度,有望成为解决肝功能替代疗法中细胞来源问题的新希望.     

The chromosome make-up of mouse embryonic stem cells is predictive of somatic and germ cell chimaerism

Mouse pluripotent embryonic stem (ES) cells, once reintroduced into a mouse blastocyst, can contribute to the formation of all tissues, including the germline, of an organism referred to as a chimaeric. However, the reasons why this contribution often appears erratic are poorly understood. We have tested the notion that the chromosome make-up may be important in contributing both to somatic cell chimaerism and to germ line transmission. We found that the percentage of chimaerism of ES cell-embryo chimaeras, the absolute number of chimaeras and the ratio of chimaeras to total pups born all correlate closely with the percentage of euploid metaphases in the ES cell clones injected into the murine blastocyst. The majority of the ES cell clones that we tested, which were obtained from different gene targeting knockout experiments and harboured 50 to 100% euploid metaphases, did transmit to the germline; in contrast, none of the ES cell clones with more than 50% of chromosomally abnormal metaphases transmitted to the germline. Euploid ES cell clones cultured in vitro for more than 20 passages rapidly became severely aneuploid, and again this correlated closely with the percentage of chimaerism and with the number of ES cell--embryo chimaeras obtained per number of blastocysts injected. At the same time, the ability of these clones to contribute to the germline was lost when the proportion of euploid cells dropped below 50%. This study suggests that aneuploidy, rather than loss of totipotency, in ES cells, is the major cause of failure in obtaining contributions to all tissues of the adult chimaera, including the germline. Because euploidy is predictive of germline transmission, karyotype analysis is crucial and time/cost saving in any gene-targeting experiment     

用于胚胎干细胞分离培养的滋养层细胞株的建立

具有发育全能性的胚干细胞在分离培养时必须依赖滋养层细胞~［１］,本研究旨在建议一个可供使用的滋养层细胞株。将小鼠胎仔去头、内脏、四肢后用胰酶消化,用作成纤维细胞的初代培养,２～３６后继代培养,并开始进行选择和株化,获得的继代培养的成纤维细胞可用于制备胚干细胞培养所需滋养层,亦可冷冻保存备用。本实验所建立的小鼠成纤维细胞系,已成功地用于胚干细胞的分离培养,证明可作为分离培养胚干细胞时所需的滋养层细胞来源。     

Efficient gene silencing and cell differentiation using siRNA in mouse and monkey ES cells

Small interfering RNA (siRNA) has been widely used for suppressing gene expression in various organisms. Here, we describe efficient methods to suppress target genes (EGFP or Oct4) using siRNA in mouse and monkey ES cells, and differentiation. In mouse ES cells, FACS analysis revealed that EGFP expression was suppressed in 97% of transfected cells at 48 h after transfection. In addition, cells expressed Hand1 and Cdx2, which are the marker genes of trophoblast lineage by the transient suppression of Oct4. In the case of monkey ES cells, highly efficient suppression was achieved in 98% of cells at 96 h post-transfection using the Sendai virus (hemagglutinating virus of Japan, HVJ) envelope as a carrier of siRNA. These efficient transfection methods using synthetic siRNA should contribute to evaluate specific gene function in ES cells and can be used to differentiate ES cells into desired cell lineages.     

At most three ES cells contribute to the somatic lineages of chimeric mice and of mice produced by ES-tetraploid complementation

Chimeric or entirely embryonic stem (ES) cell-derived mice ("ES mice") can be produced by injecting ES cells into diploid (2n) or tetraploid (4n) host blastocysts, respectively. Usually, between 10 and 15 ES cells are injected into the host blastocyst, but it is not clear how many of the injected cells contribute to the somatic lineages, thus serve as "founder cells" of the embryo proper. We have used genetically labeled ES cells to retrospectively determine the number of founder ES cells that generate the somatic lineages of chimeric and of ES mice. ES cell clones individually labeled with provirus were mixed in equal numbers and injected into 2n or 4n blastocysts to generate chimeric or ES mice. Southern analysis of DNA from the resulting animals indicated that the somatic lineages were most often derived from one or two and sometimes from up to three founder ES cells. The number of founder cells was independent of the total number of cells injected into the host blastocysts. Our results are consistent with the notion that constraints of the host embryo restrict the number of ES cells that can contribute to a chimeric or an ES mouse.     

A novel embryotoxic estimation method of VPA using ES cells differentiation system

Valproic acid (VPA), which has a wide range of therapeutic applications, is known as a potent teratogen that induces neural tube defects in vertebrates. Here, we have characterized the tissue-specific, embryotoxic effects of VPA on developmental processes using a novel system with differentiating mouse ES cells. Under our cultivating condition, ES cells differentiated into cardiomyocytes, although various cell types can be differentiated. VPA affected cell viability and differentiation from undifferentiated ES cells to cardiomyocytes in a dose-dependent manner. The analysis of tissue-specific markers also revealed that VPA potently inhibited mesodermal and endodermal development but promoted neuronal differentiation in a lineage-specific manner. Taking the in vivo teratogenicity of VPA into account, this assay system could be useful in predicting the degree of embryotoxicity of VPA. We, thus, propose that the in vivo embryotoxic effects of various medicines can be estimated fast and accurately using this in vitro cell differentiation system.     

In vitro differentiation of rat embryonic stem cells into functional cardiomyocytes

The recent breakthrough in the generation of rat embryonic stem cells (rESCs) opens the door to application of gene targeting to create models for the study of human diseases. In addition, the in vitro differentiation system from rESCs into derivatives of three germ layers will serve as a powerful tool and resource for the investigation of mammalian development, cell function, tissue repair, and drug discovery. However, these uses have been limited by the difficulty of in vitro differentiation. The aims of this study were to establish an in vitro differentiation system from rESCs and to investigate whether rESCs are capable of forming terminal-differentiated cardiomyocytes. Using newly established rESCs, we found that embryoid body (EB)-based method used in mouse ESC (mESC) differentiation failed to work for the serum-free cultivated rESCs. We then developed a protocol by combination of three chemical inhibitors and feeder-conditioned medium. Under this condition, rESCs formed EBs, propagated and differentiated into three embryonic germ layers. Moreover, rESC-formed EBs could differentiate into spontaneously beating cardiomyocytes after plating. Analyses of molecular, structural, and functional properties revealed that rESC-derived cardiomyocytes were similar to those derived from fetal rat hearts and mESCs. In conclusion, we successfully developed an in vitro differentiation system for rESCs through which functional myocytes were generated and displayed phenotypes of rat fetal cardiomyocytes. This unique cellular system will provide a new approach to study the early development and cardiac function, and serve as an important tool in pharmacological testing and cell therapy.     

同源重组技术研究进展

同源重组是近年来迅速发展起来的对细胞染色体基因组中某一特殊基因进行定向操作,以便借助转基因动物手段来精确研究基因结构与功能及表达调控的技术。本文对同源重组发生的分子机理、实验设计策略、打靶细胞的筛选和富集方法,近年来在这一领域中所取得的主要成就及应用前景进行了较全面的评述。     

A simple qPCR-based method to detect correct insertion of homologous targeting vectors in murine ES cells

The identification of correctly targeted embryonic stem (ES) cell clones from among the large number of random integrants that result from most selection paradigms remains an important hurdle in the generation of animals bearing homologously targeted transgenes. Given the limitations inherent to Southern blotting and standard PCR, we utilized quantitative real-time polymerase chain reaction (qPCR) to rapidly identify murine ES cell clones containing insertions at the correct genomic locus. Importantly, this approach is useful for screening ES clones from conditional/insertional “knock-in” strategies in which there is no loss of genetic material. Simple validation avoids the generation of assays prone to false negative results. In this method, probe and primer sets that span an insertion site detect and quantify the unperturbed gene relative to an irrelevant reference gene, allowing ES cell clones to be screened for loss of detection of one copy of the gene (functional loss of homozygousity (LOH)) that occurs when the normal DNA is disrupted by the insertion event. Simply stated, detected gene copy number falls from two to one in correctly targeted clones. We have utilized such easily designed and validated qPCR LOH assays to rapidly and accurately identify insertions in multiple target sites (including the Lepr and mTOR loci) in murine ES cells, in order to generate transgenic animals.