Generation of Transgenic Rats with YACs and BACs: Preparation Procedures and Integrity on Microinjected DNA.

Abstract

Abstract:The aim of the present study was to investigate differences in the methods for preparing a large DNA fragment to be used for making transgenic rats from the standpoint of transgenic production efficiency and integrity of the introduced gene. In yeast artificial chromosome (YAC) transgenesis, three methods for preparing DNA for microinjection were compared: amplification of YAC in yeast (AMP), amplification of YAC in yeast and removal of the amplification element (AMP/RE), and no amplification of the YAC in yeast (AMP -). Production efficiency per microinjected ovum with DNA by the AMP method was four times higher than that by the AMP/RE and AMP -. Based on these results, we favor the AMP method in spite of the thymidine kinase gene-induced male sterility. In bacterial artificial chromosome (BAC) transgenesis, linear DNA fragments for microinjection prepared by three kinds of purification procedures were compared: Not I digestion and CsCl gradient ultra-centrifugation (Prep. 1), CsCl gradient ultra-centrifugation, Not I digestion, gel electrophoresis, and β-agarase digestion (Prep. 2), and CsCl gradient ultra-centrifugation, Not I digestion, pulse field gel electrophoresis, and β-agarase digestion (Prep. 3). Although the efficiency of producing transgenic rats was similar with all these three DNA preparations, integration of the intact DNA fragment only occurred with the Prep. 3 procedure. We therefore favor the Prep. 3 method for preparing BAC DNA fragments. These results indicate that the method used to prepare a large DNA fragment such as YAC and BAC DNAs is important in order to produce transgenic rats with an intact transgene.

Variations in Promoter Activity Reveal a Differential Expression and Physiology of Glutamate Transporters by Glia in the Developing and Mature CNS

“…1 A). From a total of 62 potential founders, three mice were found to contain the GLAST-DsRed transgene, consistent with the low probability of intact BAC integration (Takahashi et al, 2000). Offspring from two of the founders (lines 593 and 570) exhibited high DsRed expression, whereas offspring from the third (line 573) expressed DsRed at a much lower level.…”

Establishment of an Efficient BAC Transgenesis Protocol and its Application to Functional Characterization of the Mouse Brachyury Locus

“…The integration efficiency or frequency of transgene fragmentation was not described in detail in these studies. Takahashi et al [22] compared three methods and found that integration of intact DNA occurred only when the BAC DNA was isolated by CsCl gradient centrifugation followed by linearization with restriction enzyme, PFGE separation, and β-agarase digestion. They found that four animals out of twelve transgenic founders (33.3%) possessed both ends of the BAC transgene.…”

“…When linearized with λ-terminase, the left and right arms of BAC DNA can be detected separately by PCR typing. If both arms are positive by PCR typing, it is likely that the entire BAC insert is integrated into the mouse genome without fragmentation [22]. As shown in Table 1, 29 integrations in 34 informative cases (85.3%) appeared to contain both right and left arms, suggesting that frequency of the fragmentation of the BAC DNA when integrated into the genome was not high with our BAC DNA preparations.…”

“…Although BAC DNA can be easily isolated by conventional plasmid preparation protocol for analytical work, care must be taken in preparing intact large DNA inserts for microinjection [5,22,24]. Takahashi et al [22] compared three purification procedures for BAC DNA, and found that transgenic animals with intact BAC inserts were obtained only with BAC isolated by CsCl gradient ultra-centrifugation followed by linearization with restriction enzyme, pulsed field gel electrophoresis (PFGE) separation, and β-agarase digestion.…”

“…Takahashi et al [22] compared three purification procedures for BAC DNA, and found that transgenic animals with intact BAC inserts were obtained only with BAC isolated by CsCl gradient ultra-centrifugation followed by linearization with restriction enzyme, pulsed field gel electrophoresis (PFGE) separation, and β-agarase digestion. We devised a similar but simpler procedure for BAC DNA isolation adopted from a YAC DNA purification method [17], and have generated 43 founder transgenic mice.…”