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A Method for High Efficiency YAC Lipofection into Murine Embryonic Stem Cells

A Method for High Efficiency YAC Lipofection into Murine Embryonic Stem Cells 5054–5055 Nucleic Acids Research, 1996, Vol. 24, No. 24  1996 Oxford University Press A method for high efficiency YAC lipofection into murine embryonic stem cells 1,2, 1 Jeannie T. Lee * and Rudolf Jaenisch Whitehead Institute for Biomedical Research, Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02142, USA and Pathology Department, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA Received September 11, 1996; Revised and Accepted November 5, 1996 ABSTRACT recovery. This previous protocol as well as other published protocols typically used 10–100 μg of purified YAC DNA and required We describe a modified protocol for introducing yeast screening of many lipofectants to obtain clones containing ‘intact’ artificial chromosomes (YACs) into murine embryonic transgenes as judged by presence of flanking vector markers and stem (ES) cells by lipofection. With a decreased insert DNA (6,10). In our modified protocol, the requirement for DNA:cell ratio, increased concentration of condensing YAC DNA was reduced 10–100-fold, the recovery of neomycin- agents and altered culture conditions, this protocol resistant colonies was increased, and the yield of clones containing reduces the requirement for YAC DNA to a few both flanking vector markers and insert DNA was greater. micrograms, improves the recovery of neomycin- The basic protocol is as follows. resistant ES colonies and increases the yield of clones (i) YAC DNA preparation: high quality, high molecular weight containing both flanking vector markers and insert. DNA was prepared by isolation from pulsed field gels. Yeast These modifications enable generation of sufficient cultures were grown to stationary phase, harvested, washed once ‘intact’ transgenic clones for biological analysis with a with 1 M sorbitol, and resuspended in 1 M Sorbitol, 0.2 M single experiment. Na HPO /NaH PO pH 7.4, 1 mM EDTA, 58 mM 2-mercapto- 2 4 2 4 Large DNA fragments carried on yeast artificial chromosomes 9 ethanol at 3 × 10 cells/ml. Cells were spheroplasted in 0.75 mg/ml (YACs) have been introduced into the mammalian genome (1,2). zymolyase 100T (ICN) at 37C for 30 min. Spheroplasts were Because YACs enable cloning of hundreds of kilobases of DNA mixed with SeaPlaque low-gelling temperature agarose (FMC) to (3), YAC transgenes offer several advantages over traditional 9 a final concentration of 1% agarose and 1 × 10 cells/ml and cast ‘minigene’ constructs. First, inclusion of remote cis-acting in BioRad DNA plug molds. DNA plugs were then digested regulatory elements can preserve temporal, spatial and dosage overnight with 0.5 mg/ml proteinase K in 0.5 M EDTA, 10 mM information encoded at the endogenous locus (4–6). Second, Tris pH 7.5 and 1% N-lauroylsarcosine, and dialyzed twice in YACs allow for the study of large genomic structures or regions 10 mM Tris pH 7.5, 20 mM EDTA, and stored in 10 mM Tris pH such as alpha-centroid DNA and the mammalian X-chromosome 7.5, 1 mM EDTA at 4C. Genomic DNA embedded in agarose inactivation center (7,8). Third, YAC templates are easy to was subjected to pulsed field electrophoresis (BioRad CHEF genetically manipulate in yeast, thus enabling rapid generation of DRII system) to separate chromosomes in the 200–1000 kb point mutation and deletions. Finally, YAC transgenic cell lines or range. With ethidium bromide-stained markers as a guide, the mice can be used as bioreactors in the production of biotherapeutics unstained gel corresponding to the desired YAC DNA was such as immunoglobulins (9,10). For all these reasons, YAC excised and dialyzed twice in 10 mM Tris pH 7.5, 1 mM EDTA, transgenics have become valuable tools in creating mouse models 200 μM spermine (Sigma) and 25 mM NaCl at room temperature for disease and vertebrate development. for 0.5 h. Using this procedure, 1–2 μg of YAC DNA have been To date, transfers have been achieved by lipofection into ES routinely recovered from 1 ml of genomic DNA (less DNA if YAC cells, by microinjection into mouse oocytes and by spheroplast is large and/or unstable). fusion with ES cells. While spheroplast fusions have reliably (ii) Embryonic stem (ES) cell lipofection: in preparation for yielded large numbers of transfectants with intact YAC transgenes, lipofection, 1 ml agarose blocks containing 300 ng of purified the incorporation of endogenous yeast genomic DNA potentially YAC DNA were combined with 4 μg/ml low-molecular weight complicates intrepretation of results. Because lipofection and poly-L-lysine (Sigma) and then melted at 68C for 10 min and microinjection use purified YAC DNA, these methodologies may digested with 1 U β-agarase (New England Biolabs) per 100 μl be preferrable for many investigators; however, the requirement block according to the manufacturer’s specification. In the author’s for large quantities of high molecular weight DNA, variability in experience, incomplete agarose digestion significantly reduced transfer efficiency and high frequency of transgene deletion have lipofection efficiency. Thus, agarasing should be allowed to been major obstacles to using these techniques in many laboratories. Here, we describe modifications of the protocol by Strauss et al. proceed at least 4 h. From this point on, all manipulation of DNA (6) which greatly simplify YAC transfer while enhancing lipofectant was done with a wide-bore pipette tip to reduce mechanical shear. *To whom correspondence should be addressed at: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02138, USA. Tel: +1 617 258 5207; Fax: +1 617 258 6505; Email: [email protected] 5055 Nucleic Acids Research, 1996, Vol. 24, No. 24 5055 Nucleic Acids Research, 1994, Vol. 22, No. 1 Table 1. Comparison of results achieved by various protocols for introducing YACs into ES cells YAC, size Input DNA Lipofection efficiency: No. clones with L & R Combined yield: no. (reference) (μg) YAC-containing YAC arms (‘intact’) ‘intact’ clones/100 μg NeoR colonies/μg modified protocol: Y116, 450 kb (8) 1.5 7 5 out of 14 (35%) 330 750 kb YAC (J.T. Lee, unpublished) 2 4.5 6 out of 9 (66%) 200–300 300 kb YAC (A.Chess, unpublished) 1.5 8 2 out of 12 (16%) 100–150 Y22, 150 kb (6) 40–100 0.5–1.0 7 out of 35 (20%) 7–14 APP-8, 650 kb (4) 0.1 20–30 3 out of 23 (13%) 200–300 J1.3P, 85 kb (10) 8 15 3 out of 15 (20%) 40 Because of smearing of YAC DNA in the 500–900 kb range in the preparative pulsed field gel, this YAC was concentrated 2–3-fold by the Amicon Centriprep-100 system at 500 g prior to lipofection. This was a co-lipofection of 8 μg of YAC DNA and 4–8-fold molar excess of a neomycin resistance plasmid. 1221 neomycin-resistant colonies were screened and 15 were found to contain J1.3P DNA. Cationic lipid DOTAP (BoehringerMannheim) (30 μg) was added, both TRP1 and NEO as well as insert DNA. This protocol is being mixed with a pipette, and incubated at room temperature for 45 min, used presently by other laboratories. In one independent laboratory, after which 1/10 vol of 10× OptiMEM (Life Technologies) was a 300 kb YAC containing olfactory receptor loci was introduced into added. The DNA complex was then mixed gently with 2 ml of cell ES cells with a similar success rate, with a combined yield of 7 + + + suspension containing 10 dispersed ES cells prepared as follows. 100–150 NEO TRP1 insert clones per 100 μg input DNA ES cells were grown in standard ES media (DME, 15% fetal bovine (Andrew Chess, Whitehead Institute, personal communication). serum, 0.1 mM amino acids and 0.1 mM β-mercaptoethanol, 500 Transgenic ES lines generated with this protocol have contributed U/ml LIF) to 70% confluence, dispersed by trypsinization, and extensively to chimeras, including to the germline (8; J. T. Lee, resuspended in 1× OptiMEM supplemented with 1000 U/ml LIF. unpublished work; A. Chess, personal communication). The DNA–cellular mixture was incubated at 37C for 5 h without Table 1 summarizes the results achieved with our protocol disturbance in a tissue culture incubator. DNA-treated ES cells were compared with those of others. Since the rate-limiting step for then combined with 10 ml of standard ES medium and plated on day successful lipofection is purification of high molecular weight DNA, 14 embryonic feeder cells. Drug selection was begun at 24 h and the the overall efficiency is normalized to amount of input DNA used. media changed daily thereafter. Drug-resistant lipofectants became Note that our modified protocol yielded a lipofection efficiency visible as colonies between days 8 and 18 after selection. 20-fold over that of Strauss et al. (6). Furthermore, a workable We believe that significant modifications in our protocol include number of ‘intact’ clones can be generated in a single experiment the use of high concentration LIF during ES cell lipofection (protocols requiring 10–100 μg input YAC DNA require many days (maintaining cells in the undifferentiated state), the use of serum- of lipofection). Since ‘intact clones’ have represented 35–66% of less OptiMEM, lower concentration of input YAC DNA, and clones generated, our protocol potentially expedites isolation of proportionally more condensing agent (spermine, poly-L-lysine). ES transgenic lines for biological analysis. In our experience, transgene cells lipofected with no LIF or lesser amounts did not look as healthy copy numbers from 1 to >20. Analysis to date indicates that as those with high LIF concentration (ES cells began to lipofection yields single integration sites at random loci in the differentiate). The use of OptiMEM instead of DMEM in the DNA genome. These sites have included pericentromeric, subtelomeric mixture also appeared to improve ES cell morphology following and central regions of at least six distinct chromosomes (J. Lee, lipofection. Notably, the use of more condensing reagents resulted unpublished observations). in formation of visible precipitates in the DNA–cell mixture, ACKNOWLEDGEMENTS although this did not seem to negatively affect lipofection. This protocol has been used successfully to introduce YAC 116, We thank Andrew Chess for sharing and discussing results prior a 450 kb clone containing the murine X-inactivation center (8; Table to publication. This work was supported by the Howard Hughes 1). In a typical experiment using 1.5 μg of purified YAC DNA, 14 Postdoctoral Fellowship for Physicians granted to J.T.L., by the neomycin-resistant colonies were obtained from lipofecting 5 × 10 Pathology Department at the Massachusetts General Hospital cells. Pulsed field and conventional Southern analyses indicated that where J.T.L. is a fellow, and by a grant from the National Institutes five clones contained both vector arms, NEO (retrofitted into the of Health/National Cancer Institute (R35-CA44339) to R.J. URA3 arm) and TRP1, as well as intact copies of the Xist gene. As published elsewhere (8), these clones also contain all necessary elements required for appropriate timing and level of Xist express- REFERENCES ion. An additional two clones contained truncations that retained the 1 Capecchi,M.R. (1993) Nature, 362, 205–206. neomycin arm and Xist insert DNA but deleted the TRP1 vector 2 Lamb,B.T. and Gearhart,J.D. (1995) Curr. Opin. Genet. Dev., 5, 342–348. arm. The remaining seven clones sustained deletions of a significant 3 Burke,D. et al. (1987) Science, 236, 806–812. amount of insert DNA. 4Lamb,B.T. et al. (1993) Nature Genet., 5, 22–30. We have also successfully introduced a second X-linked YAC of 5 Schedl,A. et al. (1993) Nature, 362, 258–261. 6 Strauss,W.M. et al. (1993) Science, 259, 1904–1907 750 kb into ES cells. While larger YAC size was expected to 7 Larin,Z. et al. (1994) Hum. Mol. Genet., 3, 689–695. negatively influence the success rate, our work with the 750 kb clone 8 Lee,J.T. et al. (1996) Cell, 86, 73–84. yielded surprisingly comparable results (Table 1). Not only was the 9 Green,L.L. et al. (1994) Nature Genet., 7, 13–21. lipofection efficiency high, but 66% of clones analyzed contained 10 Choi,T.K. et al. (1993) Nature Genet., 4, 117–123. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Nucleic Acids Research Oxford University Press

A Method for High Efficiency YAC Lipofection into Murine Embryonic Stem Cells

Lee, Jeannie T.; Jaenisch, Rudolf
Nucleic Acids Research , Volume 24 (24) – Dec 1, 1996
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Oxford University Press
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© 1996 Oxford University Press
ISSN
0305-1048
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1362-4962
DOI
10.1093/nar/24.24.5054
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Abstract

5054–5055 Nucleic Acids Research, 1996, Vol. 24, No. 24  1996 Oxford University Press A method for high efficiency YAC lipofection into murine embryonic stem cells 1,2, 1 Jeannie T. Lee * and Rudolf Jaenisch Whitehead Institute for Biomedical Research, Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02142, USA and Pathology Department, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA Received September 11, 1996; Revised and Accepted November 5, 1996 ABSTRACT recovery. This previous protocol as well as other published protocols typically used 10–100 μg of purified YAC DNA and required We describe a modified protocol for introducing yeast screening of many lipofectants to obtain clones containing ‘intact’ artificial chromosomes (YACs) into murine embryonic transgenes as judged by presence of flanking vector markers and stem (ES) cells by lipofection. With a decreased insert DNA (6,10). In our modified protocol, the requirement for DNA:cell ratio, increased concentration of condensing YAC DNA was reduced 10–100-fold, the recovery of neomycin- agents and altered culture conditions, this protocol resistant colonies was increased, and the yield of clones containing reduces the requirement for YAC DNA to a few both flanking vector markers and insert DNA was greater. micrograms, improves the recovery of neomycin- The basic protocol is as follows. resistant ES colonies and increases the yield of clones (i) YAC DNA preparation: high quality, high molecular weight containing both flanking vector markers and insert. DNA was prepared by isolation from pulsed field gels. Yeast These modifications enable generation of sufficient cultures were grown to stationary phase, harvested, washed once ‘intact’ transgenic clones for biological analysis with a with 1 M sorbitol, and resuspended in 1 M Sorbitol, 0.2 M single experiment. Na HPO /NaH PO pH 7.4, 1 mM EDTA, 58 mM 2-mercapto- 2 4 2 4 Large DNA fragments carried on yeast artificial chromosomes 9 ethanol at 3 × 10 cells/ml. Cells were spheroplasted in 0.75 mg/ml (YACs) have been introduced into the mammalian genome (1,2). zymolyase 100T (ICN) at 37C for 30 min. Spheroplasts were Because YACs enable cloning of hundreds of kilobases of DNA mixed with SeaPlaque low-gelling temperature agarose (FMC) to (3), YAC transgenes offer several advantages over traditional 9 a final concentration of 1% agarose and 1 × 10 cells/ml and cast ‘minigene’ constructs. First, inclusion of remote cis-acting in BioRad DNA plug molds. DNA plugs were then digested regulatory elements can preserve temporal, spatial and dosage overnight with 0.5 mg/ml proteinase K in 0.5 M EDTA, 10 mM information encoded at the endogenous locus (4–6). Second, Tris pH 7.5 and 1% N-lauroylsarcosine, and dialyzed twice in YACs allow for the study of large genomic structures or regions 10 mM Tris pH 7.5, 20 mM EDTA, and stored in 10 mM Tris pH such as alpha-centroid DNA and the mammalian X-chromosome 7.5, 1 mM EDTA at 4C. Genomic DNA embedded in agarose inactivation center (7,8). Third, YAC templates are easy to was subjected to pulsed field electrophoresis (BioRad CHEF genetically manipulate in yeast, thus enabling rapid generation of DRII system) to separate chromosomes in the 200–1000 kb point mutation and deletions. Finally, YAC transgenic cell lines or range. With ethidium bromide-stained markers as a guide, the mice can be used as bioreactors in the production of biotherapeutics unstained gel corresponding to the desired YAC DNA was such as immunoglobulins (9,10). For all these reasons, YAC excised and dialyzed twice in 10 mM Tris pH 7.5, 1 mM EDTA, transgenics have become valuable tools in creating mouse models 200 μM spermine (Sigma) and 25 mM NaCl at room temperature for disease and vertebrate development. for 0.5 h. Using this procedure, 1–2 μg of YAC DNA have been To date, transfers have been achieved by lipofection into ES routinely recovered from 1 ml of genomic DNA (less DNA if YAC cells, by microinjection into mouse oocytes and by spheroplast is large and/or unstable). fusion with ES cells. While spheroplast fusions have reliably (ii) Embryonic stem (ES) cell lipofection: in preparation for yielded large numbers of transfectants with intact YAC transgenes, lipofection, 1 ml agarose blocks containing 300 ng of purified the incorporation of endogenous yeast genomic DNA potentially YAC DNA were combined with 4 μg/ml low-molecular weight complicates intrepretation of results. Because lipofection and poly-L-lysine (Sigma) and then melted at 68C for 10 min and microinjection use purified YAC DNA, these methodologies may digested with 1 U β-agarase (New England Biolabs) per 100 μl be preferrable for many investigators; however, the requirement block according to the manufacturer’s specification. In the author’s for large quantities of high molecular weight DNA, variability in experience, incomplete agarose digestion significantly reduced transfer efficiency and high frequency of transgene deletion have lipofection efficiency. Thus, agarasing should be allowed to been major obstacles to using these techniques in many laboratories. Here, we describe modifications of the protocol by Strauss et al. proceed at least 4 h. From this point on, all manipulation of DNA (6) which greatly simplify YAC transfer while enhancing lipofectant was done with a wide-bore pipette tip to reduce mechanical shear. *To whom correspondence should be addressed at: Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02138, USA. Tel: +1 617 258 5207; Fax: +1 617 258 6505; Email: [email protected] 5055 Nucleic Acids Research, 1996, Vol. 24, No. 24 5055 Nucleic Acids Research, 1994, Vol. 22, No. 1 Table 1. Comparison of results achieved by various protocols for introducing YACs into ES cells YAC, size Input DNA Lipofection efficiency: No. clones with L & R Combined yield: no. (reference) (μg) YAC-containing YAC arms (‘intact’) ‘intact’ clones/100 μg NeoR colonies/μg modified protocol: Y116, 450 kb (8) 1.5 7 5 out of 14 (35%) 330 750 kb YAC (J.T. Lee, unpublished) 2 4.5 6 out of 9 (66%) 200–300 300 kb YAC (A.Chess, unpublished) 1.5 8 2 out of 12 (16%) 100–150 Y22, 150 kb (6) 40–100 0.5–1.0 7 out of 35 (20%) 7–14 APP-8, 650 kb (4) 0.1 20–30 3 out of 23 (13%) 200–300 J1.3P, 85 kb (10) 8 15 3 out of 15 (20%) 40 Because of smearing of YAC DNA in the 500–900 kb range in the preparative pulsed field gel, this YAC was concentrated 2–3-fold by the Amicon Centriprep-100 system at 500 g prior to lipofection. This was a co-lipofection of 8 μg of YAC DNA and 4–8-fold molar excess of a neomycin resistance plasmid. 1221 neomycin-resistant colonies were screened and 15 were found to contain J1.3P DNA. Cationic lipid DOTAP (BoehringerMannheim) (30 μg) was added, both TRP1 and NEO as well as insert DNA. This protocol is being mixed with a pipette, and incubated at room temperature for 45 min, used presently by other laboratories. In one independent laboratory, after which 1/10 vol of 10× OptiMEM (Life Technologies) was a 300 kb YAC containing olfactory receptor loci was introduced into added. The DNA complex was then mixed gently with 2 ml of cell ES cells with a similar success rate, with a combined yield of 7 + + + suspension containing 10 dispersed ES cells prepared as follows. 100–150 NEO TRP1 insert clones per 100 μg input DNA ES cells were grown in standard ES media (DME, 15% fetal bovine (Andrew Chess, Whitehead Institute, personal communication). serum, 0.1 mM amino acids and 0.1 mM β-mercaptoethanol, 500 Transgenic ES lines generated with this protocol have contributed U/ml LIF) to 70% confluence, dispersed by trypsinization, and extensively to chimeras, including to the germline (8; J. T. Lee, resuspended in 1× OptiMEM supplemented with 1000 U/ml LIF. unpublished work; A. Chess, personal communication). The DNA–cellular mixture was incubated at 37C for 5 h without Table 1 summarizes the results achieved with our protocol disturbance in a tissue culture incubator. DNA-treated ES cells were compared with those of others. Since the rate-limiting step for then combined with 10 ml of standard ES medium and plated on day successful lipofection is purification of high molecular weight DNA, 14 embryonic feeder cells. Drug selection was begun at 24 h and the the overall efficiency is normalized to amount of input DNA used. media changed daily thereafter. Drug-resistant lipofectants became Note that our modified protocol yielded a lipofection efficiency visible as colonies between days 8 and 18 after selection. 20-fold over that of Strauss et al. (6). Furthermore, a workable We believe that significant modifications in our protocol include number of ‘intact’ clones can be generated in a single experiment the use of high concentration LIF during ES cell lipofection (protocols requiring 10–100 μg input YAC DNA require many days (maintaining cells in the undifferentiated state), the use of serum- of lipofection). Since ‘intact clones’ have represented 35–66% of less OptiMEM, lower concentration of input YAC DNA, and clones generated, our protocol potentially expedites isolation of proportionally more condensing agent (spermine, poly-L-lysine). ES transgenic lines for biological analysis. In our experience, transgene cells lipofected with no LIF or lesser amounts did not look as healthy copy numbers from 1 to >20. Analysis to date indicates that as those with high LIF concentration (ES cells began to lipofection yields single integration sites at random loci in the differentiate). The use of OptiMEM instead of DMEM in the DNA genome. These sites have included pericentromeric, subtelomeric mixture also appeared to improve ES cell morphology following and central regions of at least six distinct chromosomes (J. Lee, lipofection. Notably, the use of more condensing reagents resulted unpublished observations). in formation of visible precipitates in the DNA–cell mixture, ACKNOWLEDGEMENTS although this did not seem to negatively affect lipofection. This protocol has been used successfully to introduce YAC 116, We thank Andrew Chess for sharing and discussing results prior a 450 kb clone containing the murine X-inactivation center (8; Table to publication. This work was supported by the Howard Hughes 1). In a typical experiment using 1.5 μg of purified YAC DNA, 14 Postdoctoral Fellowship for Physicians granted to J.T.L., by the neomycin-resistant colonies were obtained from lipofecting 5 × 10 Pathology Department at the Massachusetts General Hospital cells. Pulsed field and conventional Southern analyses indicated that where J.T.L. is a fellow, and by a grant from the National Institutes five clones contained both vector arms, NEO (retrofitted into the of Health/National Cancer Institute (R35-CA44339) to R.J. URA3 arm) and TRP1, as well as intact copies of the Xist gene. As published elsewhere (8), these clones also contain all necessary elements required for appropriate timing and level of Xist express- REFERENCES ion. An additional two clones contained truncations that retained the 1 Capecchi,M.R. (1993) Nature, 362, 205–206. neomycin arm and Xist insert DNA but deleted the TRP1 vector 2 Lamb,B.T. and Gearhart,J.D. (1995) Curr. Opin. Genet. Dev., 5, 342–348. arm. The remaining seven clones sustained deletions of a significant 3 Burke,D. et al. (1987) Science, 236, 806–812. amount of insert DNA. 4Lamb,B.T. et al. (1993) Nature Genet., 5, 22–30. We have also successfully introduced a second X-linked YAC of 5 Schedl,A. et al. (1993) Nature, 362, 258–261. 6 Strauss,W.M. et al. (1993) Science, 259, 1904–1907 750 kb into ES cells. While larger YAC size was expected to 7 Larin,Z. et al. (1994) Hum. Mol. Genet., 3, 689–695. negatively influence the success rate, our work with the 750 kb clone 8 Lee,J.T. et al. (1996) Cell, 86, 73–84. yielded surprisingly comparable results (Table 1). Not only was the 9 Green,L.L. et al. (1994) Nature Genet., 7, 13–21. lipofection efficiency high, but 66% of clones analyzed contained 10 Choi,T.K. et al. (1993) Nature Genet., 4, 117–123.

Journal

Nucleic Acids Research – Oxford University Press

Published: Dec 1, 1996

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