BAC DNA transgenesis requires the purification of intact BAC DNA. Birth rates and transgenic rates are dependent on carefully calibrated concentrations of microinjected DNA (). The use of polyamine microinjection buffer protects BAC DNA from fragmentation and does not interfere with in vitro
development of microinjected eggs ( and , ). Transgenesis efficiency is independent of the BAC size, BAC DNA purification method, and form (linear or circular) of BAC molecules that are microinjected (). Transgenic founders that express genes contained in BACs are generated for the majority of BAC transgenes. Genes in some BACs were not expressed, perhaps because of missing regulatory elements in genes that are too large to be contained in a single BAC or the absence of a complete genetic context. Multiple genotyping markers show that it is not unusual to generate transgenic founders that contain intact BAC molecules and founders that contain BAC fragments from the same BAC transgene (-). Complete BACs are more likely to provide physiologically relevant expression patterns and copy dependent expression levels while the examination of fragmented BACs may be useful to identify regulatory DNA sequences (Deal et al. 2006
, Dunnick et al. 2004
, Dunnick et al. 2005
, Krebs et al. 2003
, Lehoczky et al. 2008
, Sun et al. 2003
). An important key to success in BAC transgenesis is to control microinjection DNA concentrations so that the number of pups born and the number of transgenic founders generated are balanced.
Identification of a BAC DNA concentration that provides reasonable post-microinjection survival rates, birth rates, and transgenic rates is crucial. Eggs injected with BAC DNA molecules are more sensitive to the toxicity of high DNA concentrations than those injected with high concentrations of small plasmid transgenes. For example, when BAC 1282 was microinjected at 1 ng/ul we observed an extremely low birth rate (10 pups born from 443 microinjected eggs). Upon 10 fold dilution of the injection concentration to 0.1 ng/ul the birth rate improved to 107 pups from 730 injected eggs, but only two transgenic founders were identified. An ideal concentration would balance birth rates and transgenic rates more evenly. Compared to small plasmid transgenes, which are microinjected at 1-2 ng/ul (Brinster et al. 1985
), the concentration range for BACs is narrower and can vary among BAC DNA preparations. The molarity of small plasmid transgene molecules results in the injection of a few hundred transgene molecules per mouse egg. During BAC microinjection 10 to 100 fold fewer DNA molecules are introduced per egg. The percentage of transgenic pups born from the microinjection of large DNA transgenes is comparable to the percentage obtained from small DNA microinjection (Schedl et al. 1993b
; Callow et al. 1994
; Antoch et al. 1997
; Probst et al. 1998
). As many as 1000 copies of small transgenes can integrate into a single chromosomal site in transgenic mice (Lo 1986
). In BAC transgenic mice, the majority of BAC transgene concatemers contain fewer than a dozen copies (Camper and Saunders 2000
; Giraldo and Montoliu 2001
), although integration of a 76-copy BAC concatemer has been reported (Chandler et al. 2007
). The lower copy numbers typically observed in BAC transgenesis most likely reflects the smaller number of DNA molecules microinjected into each fertilized egg. Estimates of a few hundred plasmid molecules microinjected per fertilized mouse egg (Brinster et al. 1985
) are consistent with the microinjection of a few tens of BAC molecules per egg. Even though fewer molecules are microinjected, BAC transgenic efficiency is comparable to that of smaller plasmid transgenes.
High BAC DNA concentrations result in viscous DNA solutions that can be observed as they flow out of injection needles and mix with the media in the microinjection chamber. Such sticky DNA preparations trap nucleoli upon pronuclear injection and pull them out of the egg as the microinjection needle is removed. This results in egg lysis, which reduces the number of viable pups that can be obtained. We use a concentration of 0.5 ng/ul is for the initial microinjection session. This is adjusted downward to a lower concentration if one of the following conditions is noted: high viscosity of the microinjection solution causing difficulty with the injections (“sticky” needles); an extraordinarily high rate of lysis of injected eggs during the injection session (less than 70% survival); a low 2-cell embryo rate for injected eggs allowed to culture overnight (less than 70%), or an unusually low birth rate for pups (less than 10%). If a high birth rate, accompanied by a low transgenic rate is the result, then the DNA is reinjected at a higher concentration. On those occasions when BAC transgenic mice are not produced in the first microinjection sessions, the most practical solution is to purify and microinject a new BAC DNA preparation.
Examination of transgenic efficiency (the number of transgenic founders produced per microinjected eggs) facilitates comparisons between technical methods. In this context the purification methods of ion exchange, column chromatography, or CsCl gradients are equivalent (). Differences in transgenic efficiency between linear and circular BAC DNA fragments were not observed (reviewed in Camper and Saunders 2000
), unlike those differences reported for linear and supercoiled small plasmid transgenes (Brinster et al. 1985
). Systematic interference with expression by the plasmid backbone of the BAC cloning vector was not observed, although this was reported for plasmid transgenes (Hammer et al. 1987
; Kjer-Nielsen et al. 1992
; Townes et al. 1985
In transgenesis with circular BAC DNA it is sometimes assumed that random breaks will occur in the BAC, interrupt the gene of interest (particularly if the gene occupies most of the BAC), and preclude gene expression. This intuitive assumption about transgene DNA integration is incongruent with existing evidence and models for exogenous DNA insertion into chromosomes in fertilized eggs. Integration of experimentally introduced DNA in eggs occurs after a process of homologous recombination between transgene molecules to form a tandem array (Luciw et al. 1983
; Wagner et al. 1996
; reviewed by Bishop 1996
). The process of recombination resolves DNA fragments into intact molecules (Fiorenza et al. 2001
). Some research groups have taken advantage of this process to build larger transgenes from smaller DNA molecules, including plasmid (Keegan et al. 1994
), cosmid (Tacken et al. 2001
) and P1 clones (Wagner et al. 1996
). There is no a priori
advantage for the use of linear BAC DNA since both circular BACs and linear fragments mediate transgenesis efficiently with the same expression outcomes.
Either circular or linearized BAC DNA can be used for pronuclear microinjection. Thus, circular BAC DNA most commonly is microinjected since it is simpler to purify. Transgenic efficiency is the same among BAC DNA molecules ranging in size from 61 to 303 kb. Circular BAC DNA is conveniently isolated from bacterial cultures with available commercial kits. Careful DNA preparation and the use of polyamine microinjection buffer protect circular BACs from shearing. The isolation of linearized BACs free of the cloning vector backbone or the isolation of subfragments requires additional manipulations that reduce DNA yields and risk DNA shearing. Unless there is a cogent reason for BAC DNA fragment purification, such as the analysis of gene regulatory regions or the exclusion of genes from multigenic BACs, the preparation of transgenic mice with circular BAC DNA molecules is warranted.
The desired outcome of BAC transgenesis is the expression of genes contained within BACs. Because of their size, it is advantageous to genotype BAC transgenic mice with multiple markers across the BAC. Transgenic mouse lines positive for two or more markers contained in the BAC were more than twice as likely to express the gene of interest as lines positive for only one marker (-). Two-thirds of BACs for which multiple markers were available produced transgenic lines that contained BAC fragments in addition to lines that contained intact BACs (-). Genotyping transgenic founders with multiple markers is essential for the identification of genes and regulatory elements that confer gene expression in the founders. Overall 80% of BACs produced transgenic lines that expressed the desired genes. Despite the need to differentiate between intact and incomplete BAC integrations, the analysis of partial BAC transgenes can be advantageous for the identification of genes in positional cloning projects (Antoch et al. 1997
; Jones et al. 2003
) or the identification of gene regulatory elements (Deal et al. 2006
Ion Exchange BAC DNA Purification and Expression: Transgenic Founders Identified with One Genetic Marker Internal to BAC Transgene.
Size Exclusion BAC DNA Purification and Expression: Transgenic Founders Identified with a Single Genetic Marker Unique to the BAC Transgene.
Production of transgenic mice with large transgenes is more difficult due to stringent requirements for highly purified intact DNA molecules. Multiple genotyping markers across BAC transgenes are essential to verify that intact BAC molecules integrate in transgenic founders. Compared to plasmid DNA molecules the DNA concentration for effective transgenesis falls into a more narrow range for BACs than for plasmid transgenes. Similarly, the optimum BAC DNA transgenesis conditions that provide good post-injection survival rates, reasonable birth rates, and effective transgenic rates are more difficult to establish than with plasmid DNA transgenes.